<?xml version="1.0" encoding="utf-8"?>
<!--XBRL document created with Toppan Merrill Bridge  11.4.0.1 -->
<!--Based on XBRL 2.1-->
<!--Created on: 8/12/2026 7:24:19 AM-->
<!--Modified on: 8/12/2026 7:24:19 AM-->
<link:linkbase xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xbrli="http://www.xbrl.org/2003/instance" xmlns:xlink="http://www.w3.org/1999/xlink">
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#StatementCondensedConsolidatedStatementOfChangesInEquity" roleURI="http://www.proqr.com/role/StatementCondensedConsolidatedStatementOfChangesInEquity" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureGeneralInformationDetails" roleURI="http://www.proqr.com/role/DisclosureGeneralInformationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureCriticalAccountingEstimatesAndJudgementsDetails" roleURI="http://www.proqr.com/role/DisclosureCriticalAccountingEstimatesAndJudgementsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosurePropertyPlantAndEquipmentDetails" roleURI="http://www.proqr.com/role/DisclosurePropertyPlantAndEquipmentDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureBorrowingsDetails" roleURI="http://www.proqr.com/role/DisclosureBorrowingsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureLeaseLiabilitiesDetails" roleURI="http://www.proqr.com/role/DisclosureLeaseLiabilitiesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureDeferredIncomeDetails" roleURI="http://www.proqr.com/role/DisclosureDeferredIncomeDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureShareholdersEquityShareCapitalDetails" roleURI="http://www.proqr.com/role/DisclosureShareholdersEquityShareCapitalDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureShareholdersEquitySaleOfSharesDetails" roleURI="http://www.proqr.com/role/DisclosureShareholdersEquitySaleOfSharesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureShareholdersEquityShareOptionsDetails" roleURI="http://www.proqr.com/role/DisclosureShareholdersEquityShareOptionsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureRevenueDetails" roleURI="http://www.proqr.com/role/DisclosureRevenueDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureOtherIncomeNarrativeDetails" roleURI="http://www.proqr.com/role/DisclosureOtherIncomeNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="prqr-20260630.xsd#DisclosureInvestmentsInFinancialAssetsDetails" roleURI="http://www.proqr.com/role/DisclosureInvestmentsInFinancialAssetsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <link:arcroleRef arcroleURI="http://www.esma.europa.eu/xbrl/esef/arcrole/wider-narrower" xlink:type="simple" xlink:href="http://www.xbrl.org/lrr/arcrole/esma-arcrole-2018-11-21.xsd#wider-narrower" />
  <link:roleRef roleURI="http://www.proqr.com/role/DisclosureStandardAndCustomAxisDomainDefaults" xlink:href="prqr-20260630.xsd#DisclosureStandardAndCustomAxisDomainDefaults" xlink:type="simple" />
  <link:roleRef roleURI="http://fasb.org/us-gaap/role/eedm/ExtensibleEnumerationLists" xlink:type="simple" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-roles-2025.xsd#eedm" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" />
  <link:definitionLink xlink:type="extended" xlink:role="http://fasb.org/us-gaap/role/eedm/ExtensibleEnumerationLists" xlink:title="Extensible Enumeration Domain Members" />
  <link:definitionLink xlink:role="http://www.proqr.com/role/DisclosureStandardAndCustomAxisDomainDefaults" xlink:type="extended" xlink:title="99900 - Disclosure - Standard And Custom Axis Domain Defaults">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:title="dei_LegalEntityAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:title="dei_EntityDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:dei_LegalEntityAxis To dei_EntityDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ComponentsOfEquityAxis" xlink:label="ifrs-full_ComponentsOfEquityAxis" xlink:title="ifrs-full_ComponentsOfEquityAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ComponentsOfEquityDomain" xlink:label="ifrs-full_ComponentsOfEquityDomain" xlink:title="ifrs-full_EquityMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ComponentsOfEquityAxis" xlink:to="ifrs-full_ComponentsOfEquityDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ComponentsOfEquityAxis To ifrs-full_EquityMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CategoriesOfRelatedPartiesAxis" xlink:label="ifrs-full_CategoriesOfRelatedPartiesAxis" xlink:title="ifrs-full_CategoriesOfRelatedPartiesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CategoriesOfRelatedPartiesDomain" xlink:label="ifrs-full_CategoriesOfRelatedPartiesDomain" xlink:title="ifrs-full_EntitysTotalForRelatedPartiesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_CategoriesOfRelatedPartiesAxis" xlink:to="ifrs-full_CategoriesOfRelatedPartiesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_CategoriesOfRelatedPartiesAxis To ifrs-full_EntitysTotalForRelatedPartiesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInSubsidiariesAxis" xlink:label="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" xlink:title="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:label="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:title="ifrs-full_EntitysTotalForSubsidiariesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" xlink:to="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_SignificantInvestmentsInSubsidiariesAxis To ifrs-full_EntitysTotalForSubsidiariesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInAssociatesAxis" xlink:label="ifrs-full_SignificantInvestmentsInAssociatesAxis" xlink:title="ifrs-full_SignificantInvestmentsInAssociatesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInAssociatesDomain" xlink:label="ifrs-full_SignificantInvestmentsInAssociatesDomain" xlink:title="ifrs-full_EntitysTotalForAssociatesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_SignificantInvestmentsInAssociatesAxis" xlink:to="ifrs-full_SignificantInvestmentsInAssociatesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_SignificantInvestmentsInAssociatesAxis To ifrs-full_EntitysTotalForAssociatesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InitiallyAppliedIFRSsAxis" xlink:label="ifrs-full_InitiallyAppliedIFRSsAxis" xlink:title="ifrs-full_InitiallyAppliedIFRSsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InitiallyAppliedIFRSsDomain" xlink:label="ifrs-full_InitiallyAppliedIFRSsDomain" xlink:title="ifrs-full_InitiallyAppliedIFRSsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_InitiallyAppliedIFRSsAxis" xlink:to="ifrs-full_InitiallyAppliedIFRSsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_InitiallyAppliedIFRSsAxis To ifrs-full_InitiallyAppliedIFRSsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillAxis" xlink:label="ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillAxis" xlink:title="ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillDomain" xlink:label="ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillDomain" xlink:title="ifrs-full_IntangibleAssetsOtherThanGoodwillMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillAxis" xlink:to="ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfIntangibleAssetsOtherThanGoodwillAxis To ifrs-full_IntangibleAssetsOtherThanGoodwillMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:title="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:title="ifrs-full_PropertyPlantAndEquipmentMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:to="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis To ifrs-full_PropertyPlantAndEquipmentMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RangeAxis" xlink:label="ifrs-full_RangeAxis" xlink:title="ifrs-full_RangeAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RangeDomain" xlink:label="ifrs-full_RangeDomain" xlink:title="ifrs-full_RangesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_RangeAxis" xlink:to="ifrs-full_RangeDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_RangeAxis To ifrs-full_RangesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MaturityGroupingsAxis" xlink:label="prqr_MaturityGroupingsAxis" xlink:title="prqr_MaturityGroupingsAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MaturityGroupingsDomain" xlink:label="prqr_MaturityGroupingsDomain" xlink:title="prqr_MaturityGroupingsDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_MaturityGroupingsAxis" xlink:to="prqr_MaturityGroupingsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_MaturityGroupingsAxis To prqr_MaturityGroupingsDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CarryingAmountAccumulatedDepreciationAmortisationAndImpairmentAndGrossCarryingAmountAxis" xlink:label="ifrs-full_CarryingAmountAccumulatedDepreciationAmortisationAndImpairmentAndGrossCarryingAmountAxis" xlink:title="ifrs-full_CarryingAmountAccumulatedDepreciationAmortisationAndImpairmentAndGrossCarryingAmountAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CarryingAmountMember" xlink:label="ifrs-full_CarryingAmountMember" xlink:title="ifrs-full_CarryingAmountMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_CarryingAmountAccumulatedDepreciationAmortisationAndImpairmentAndGrossCarryingAmountAxis" xlink:to="ifrs-full_CarryingAmountMember" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_CarryingAmountAccumulatedDepreciationAmortisationAndImpairmentAndGrossCarryingAmountAxis To ifrs-full_CarryingAmountMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" xlink:label="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" xlink:title="prqr_IfrsIncomeStatementLocationAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" xlink:label="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" xlink:title="prqr_IfrsIncomeStatementLocationDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" xlink:to="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsIncomeStatementLocationAxis To prqr_IfrsIncomeStatementLocationDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_GeographicalAreasAxis" xlink:label="ifrs-full_GeographicalAreasAxis" xlink:title="ifrs-full_GeographicalAreasAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_GeographicalAreasDomain" xlink:label="ifrs-full_GeographicalAreasDomain" xlink:title="ifrs-full_GeographicalAreasMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_GeographicalAreasAxis" xlink:to="ifrs-full_GeographicalAreasDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_GeographicalAreasAxis To ifrs-full_GeographicalAreasMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_BorrowingsByNameAxis" xlink:label="ifrs-full_BorrowingsByNameAxis" xlink:title="ifrs-full_BorrowingsByNameAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_BorrowingsByNameDomain" xlink:label="ifrs-full_BorrowingsByNameDomain" xlink:title="ifrs-full_BorrowingsByNameMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_BorrowingsByNameAxis" xlink:to="ifrs-full_BorrowingsByNameDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_BorrowingsByNameAxis To ifrs-full_BorrowingsByNameMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfAssetsAxis" xlink:label="ifrs-full_ClassesOfAssetsAxis" xlink:title="ifrs-full_ClassesOfAssetsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfAssetsDomain" xlink:label="ifrs-full_ClassesOfAssetsDomain" xlink:title="ifrs-full_ClassesOfAssetsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfAssetsAxis" xlink:to="ifrs-full_ClassesOfAssetsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfAssetsAxis To ifrs-full_ClassesOfAssetsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfShareCapitalAxis" xlink:label="ifrs-full_ClassesOfShareCapitalAxis" xlink:title="ifrs-full_ClassesOfShareCapitalAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfShareCapitalDomain" xlink:label="ifrs-full_ClassesOfShareCapitalDomain" xlink:title="ifrs-full_ClassesOfShareCapitalMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfShareCapitalAxis" xlink:to="ifrs-full_ClassesOfShareCapitalDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfShareCapitalAxis To ifrs-full_ClassesOfShareCapitalMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSubsidiarySaleOfStockAxis" xlink:label="prqr_IfrsSubsidiarySaleOfStockAxis" xlink:title="prqr_IfrsSubsidiarySaleOfStockAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockNameOfTransactionDomain" xlink:label="prqr_IfrsSaleOfStockNameOfTransactionDomain" xlink:title="prqr_IfrsSaleOfStockNameOfTransactionDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsSubsidiarySaleOfStockAxis" xlink:to="prqr_IfrsSaleOfStockNameOfTransactionDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsSubsidiarySaleOfStockAxis To prqr_IfrsSaleOfStockNameOfTransactionDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsCounterpartyNameAxis" xlink:label="prqr_IfrsCounterpartyNameAxis" xlink:title="prqr_IfrsCounterpartyNameAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsRepurchaseAgreementCounterpartyNameDomain" xlink:label="prqr_IfrsRepurchaseAgreementCounterpartyNameDomain" xlink:title="prqr_IfrsRepurchaseAgreementCounterpartyNameDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsCounterpartyNameAxis" xlink:to="prqr_IfrsRepurchaseAgreementCounterpartyNameDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsCounterpartyNameAxis To prqr_IfrsRepurchaseAgreementCounterpartyNameDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockAxis" xlink:label="prqr_IfrsSaleOfStockAxis" xlink:title="prqr_IfrsSaleOfStockAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockDomain" xlink:label="prqr_IfrsSaleOfStockDomain" xlink:title="prqr_IfrsSaleOfStockDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsSaleOfStockAxis" xlink:to="prqr_IfrsSaleOfStockDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsSaleOfStockAxis To prqr_IfrsSaleOfStockDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfSharebasedPaymentArrangementsAxis" xlink:label="ifrs-full_TypesOfSharebasedPaymentArrangementsAxis" xlink:title="ifrs-full_TypesOfSharebasedPaymentArrangementsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfSharebasedPaymentArrangementsDomain" xlink:label="ifrs-full_TypesOfSharebasedPaymentArrangementsDomain" xlink:title="ifrs-full_SharebasedPaymentArrangementsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_TypesOfSharebasedPaymentArrangementsAxis" xlink:to="ifrs-full_TypesOfSharebasedPaymentArrangementsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_TypesOfSharebasedPaymentArrangementsAxis To ifrs-full_SharebasedPaymentArrangementsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" xlink:title="ifrs-full_CounterpartiesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" xlink:title="ifrs-full_CounterpartiesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_CounterpartiesAxis To ifrs-full_CounterpartiesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProductsAndServicesAxis" xlink:label="ifrs-full_ProductsAndServicesAxis" xlink:title="ifrs-full_ProductsAndServicesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProductsAndServicesDomain" xlink:label="ifrs-full_ProductsAndServicesDomain" xlink:title="ifrs-full_ProductsAndServicesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ProductsAndServicesAxis" xlink:to="ifrs-full_ProductsAndServicesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ProductsAndServicesAxis To ifrs-full_ProductsAndServicesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsBalanceSheetLocationAxis" xlink:label="prqr_IfrsBalanceSheetLocationAxis" xlink:title="prqr_IfrsBalanceSheetLocationAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsBalanceSheetLocationDomain" xlink:label="prqr_IfrsBalanceSheetLocationDomain" xlink:title="prqr_IfrsBalanceSheetLocationDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsBalanceSheetLocationAxis" xlink:to="prqr_IfrsBalanceSheetLocationDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsBalanceSheetLocationAxis To prqr_IfrsBalanceSheetLocationDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfContractsAxis" xlink:label="ifrs-full_TypesOfContractsAxis" xlink:title="ifrs-full_TypesOfContractsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfContractsDomain" xlink:label="ifrs-full_TypesOfContractsDomain" xlink:title="ifrs-full_TypesOfContractsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_TypesOfContractsAxis" xlink:to="ifrs-full_TypesOfContractsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_TypesOfContractsAxis To ifrs-full_TypesOfContractsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:title="srt_CounterpartyNameAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:title="srt_RepurchaseAgreementCounterpartyNameDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_CounterpartyNameAxis To srt_RepurchaseAgreementCounterpartyNameDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfFinancialAssetsAxis" xlink:label="ifrs-full_ClassesOfFinancialAssetsAxis" xlink:title="ifrs-full_ClassesOfFinancialAssetsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfFinancialAssetsDomain" xlink:label="ifrs-full_ClassesOfFinancialAssetsDomain" xlink:title="ifrs-full_FinancialAssetsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfFinancialAssetsAxis" xlink:to="ifrs-full_ClassesOfFinancialAssetsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfFinancialAssetsAxis To ifrs-full_FinancialAssetsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:label="srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:title="srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="srt_EquityMethodInvesteeNameDomain" xlink:title="srt_EquityMethodInvesteeNameDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="srt_EquityMethodInvesteeNameDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis To srt_EquityMethodInvesteeNameDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsScenarioAxis" xlink:label="prqr_IfrsScenarioAxis" xlink:title="prqr_IfrsScenarioAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsScenarioUnspecifiedDomain" xlink:label="prqr_IfrsScenarioUnspecifiedDomain" xlink:title="prqr_IfrsScenarioUnspecifiedDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsScenarioAxis" xlink:to="prqr_IfrsScenarioUnspecifiedDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsScenarioAxis To prqr_IfrsScenarioUnspecifiedDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NonadjustingEventsAfterReportingPeriodAxis" xlink:label="ifrs-full_NonadjustingEventsAfterReportingPeriodAxis" xlink:title="ifrs-full_NonadjustingEventsAfterReportingPeriodAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NonadjustingEventsAfterReportingPeriodDomain" xlink:label="ifrs-full_NonadjustingEventsAfterReportingPeriodDomain" xlink:title="ifrs-full_NonadjustingEventsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_NonadjustingEventsAfterReportingPeriodAxis" xlink:to="ifrs-full_NonadjustingEventsAfterReportingPeriodDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_NonadjustingEventsAfterReportingPeriodAxis To ifrs-full_NonadjustingEventsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ItemsOfContingentLiabilitiesAxis" xlink:label="ifrs-full_ItemsOfContingentLiabilitiesAxis" xlink:title="ifrs-full_ItemsOfContingentLiabilitiesAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ItemsOfContingentLiabilitiesDomain" xlink:label="ifrs-full_ItemsOfContingentLiabilitiesDomain" xlink:title="ifrs-full_ItemsOfContingentLiabilitiesMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ItemsOfContingentLiabilitiesAxis" xlink:to="ifrs-full_ItemsOfContingentLiabilitiesDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ItemsOfContingentLiabilitiesAxis To ifrs-full_ItemsOfContingentLiabilitiesMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_MaturityAxis" xlink:label="ifrs-full_MaturityAxis" xlink:title="ifrs-full_MaturityAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_MaturityDomain" xlink:label="ifrs-full_MaturityDomain" xlink:title="ifrs-full_AggregatedTimeBandsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_MaturityAxis" xlink:to="ifrs-full_MaturityDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_MaturityAxis To ifrs-full_AggregatedTimeBandsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_OwnershipAxis" xlink:label="srt_OwnershipAxis" xlink:title="srt_OwnershipAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_OwnershipDomain" xlink:label="srt_OwnershipDomain" xlink:title="srt_OwnershipDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_OwnershipAxis" xlink:to="srt_OwnershipDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_OwnershipAxis To srt_OwnershipDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSubsequentEventsAxis" xlink:label="prqr_IfrsSubsequentEventsAxis" xlink:title="prqr_IfrsSubsequentEventsAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSubsequentEventsDomain" xlink:label="prqr_IfrsSubsequentEventsDomain" xlink:title="prqr_IfrsSubsequentEventsDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsSubsequentEventsAxis" xlink:to="prqr_IfrsSubsequentEventsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsSubsequentEventsAxis To prqr_IfrsSubsequentEventsDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementScenarioAxis" xlink:label="srt_StatementScenarioAxis" xlink:title="srt_StatementScenarioAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="srt_ScenarioUnspecifiedDomain" xlink:title="srt_ScenarioUnspecifiedDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_StatementScenarioAxis" xlink:to="srt_ScenarioUnspecifiedDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_StatementScenarioAxis To srt_ScenarioUnspecifiedDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NewIFRSsAxis" xlink:label="ifrs-full_NewIFRSsAxis" xlink:title="ifrs-full_NewIFRSsAxis" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NewIFRSsDomain" xlink:label="ifrs-full_NewIFRSsDomain" xlink:title="ifrs-full_NewIFRSsMember" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_NewIFRSsAxis" xlink:to="ifrs-full_NewIFRSsDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_NewIFRSsAxis To ifrs-full_NewIFRSsMember" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStatementScenarioAxis" xlink:label="prqr_IfrsStatementScenarioAxis" xlink:title="prqr_IfrsStatementScenarioAxis" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ScenarioDomain" xlink:label="prqr_ScenarioDomain" xlink:title="prqr_ScenarioDomain" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsStatementScenarioAxis" xlink:to="prqr_ScenarioDomain" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsStatementScenarioAxis To prqr_ScenarioDomain" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfInterestRatesAxis" xlink:label="ifrs-full_TypesOfInterestRatesAxis_1" xlink:title="ifrs-full_TypesOfInterestRatesAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfInterestRatesDomain" xlink:label="ifrs-full_TypesOfInterestRatesDomain_1" xlink:title="ifrs-full_InterestRateTypesMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_TypesOfInterestRatesAxis_1" xlink:to="ifrs-full_TypesOfInterestRatesDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_TypesOfInterestRatesAxis_1 To ifrs-full_InterestRateTypesMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_PastDueStatusAxis" xlink:label="ifrs-full_PastDueStatusAxis_1" xlink:title="ifrs-full_PastDueStatusAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_PastDueStatusDomain" xlink:label="ifrs-full_PastDueStatusDomain_1" xlink:title="ifrs-full_PastDueStatusMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_PastDueStatusAxis_1" xlink:to="ifrs-full_PastDueStatusDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_PastDueStatusAxis_1 To ifrs-full_PastDueStatusMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfFinancialLiabilitiesAxis" xlink:label="ifrs-full_ClassesOfFinancialLiabilitiesAxis_1" xlink:title="ifrs-full_ClassesOfFinancialLiabilitiesAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfFinancialLiabilitiesDomain" xlink:label="ifrs-full_ClassesOfFinancialLiabilitiesDomain_1" xlink:title="ifrs-full_FinancialLiabilitiesMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_ClassesOfFinancialLiabilitiesAxis_1" xlink:to="ifrs-full_ClassesOfFinancialLiabilitiesDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_ClassesOfFinancialLiabilitiesAxis_1 To ifrs-full_FinancialLiabilitiesMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressesAddressTypeAxis" xlink:label="dei_EntityAddressesAddressTypeAxis_1" xlink:title="dei_EntityAddressesAddressTypeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AddressTypeDomain" xlink:label="dei_AddressTypeDomain_1" xlink:title="dei_AddressTypeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="dei_EntityAddressesAddressTypeAxis_1" xlink:to="dei_AddressTypeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:dei_EntityAddressesAddressTypeAxis_1 To dei_AddressTypeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis_1" xlink:title="srt_TitleOfIndividualAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" xlink:title="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_TitleOfIndividualAxis_1" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_TitleOfIndividualAxis_1 To srt_TitleOfIndividualWithRelationshipToEntityDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeAxis" xlink:label="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeAxis_1" xlink:title="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeDomain" xlink:label="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeDomain_1" xlink:title="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeAxis_1" xlink:to="prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeAxis_1 To prqr_IfrsCollaborativeArrangementAndArrangementOtherThanCollaborativeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_CurrencyAxis" xlink:label="srt_CurrencyAxis_1" xlink:title="srt_CurrencyAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/currency/2025/currency-2025.xsd#currency_AllCurrenciesDomain" xlink:label="currency_AllCurrenciesDomain_1" xlink:title="currency_AllCurrenciesDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="srt_CurrencyAxis_1" xlink:to="currency_AllCurrenciesDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:srt_CurrencyAxis_1 To currency_AllCurrenciesDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfRisksAxis" xlink:label="ifrs-full_TypesOfRisksAxis_1" xlink:title="ifrs-full_TypesOfRisksAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TypesOfRisksDomain" xlink:label="ifrs-full_TypesOfRisksDomain_1" xlink:title="ifrs-full_TypesOfRisksMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_TypesOfRisksAxis_1" xlink:to="ifrs-full_TypesOfRisksDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_TypesOfRisksAxis_1 To ifrs-full_TypesOfRisksMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RetrospectiveApplicationAndRetrospectiveRestatementAxis" xlink:label="ifrs-full_RetrospectiveApplicationAndRetrospectiveRestatementAxis_1" xlink:title="ifrs-full_RetrospectiveApplicationAndRetrospectiveRestatementAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RestatedMember" xlink:label="ifrs-full_RestatedMember_1" xlink:title="ifrs-full_RestatedMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_RetrospectiveApplicationAndRetrospectiveRestatementAxis_1" xlink:to="ifrs-full_RestatedMember_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_RetrospectiveApplicationAndRetrospectiveRestatementAxis_1 To ifrs-full_RestatedMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CategoriesOfFinancialAssetsAxis" xlink:label="ifrs-full_CategoriesOfFinancialAssetsAxis_1" xlink:title="ifrs-full_CategoriesOfFinancialAssetsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CategoriesOfFinancialAssetsDomain" xlink:label="ifrs-full_CategoriesOfFinancialAssetsDomain_1" xlink:title="ifrs-full_FinancialAssetsCategoryMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_CategoriesOfFinancialAssetsAxis_1" xlink:to="ifrs-full_CategoriesOfFinancialAssetsDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_CategoriesOfFinancialAssetsAxis_1 To ifrs-full_FinancialAssetsCategoryMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsAxis" xlink:label="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsAxis_1" xlink:title="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsDomain" xlink:label="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsDomain_1" xlink:title="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsMember_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsAxis_1" xlink:to="ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsAxis_1 To ifrs-full_TemporaryDifferenceUnusedTaxLossesAndUnusedTaxCreditsMember_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsClassesOfWarrantsOrRightsAxisAxis" xlink:label="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis_1" xlink:title="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis_1" />
    <link:loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ClassesOfWarrantsOrRightsDomain" xlink:label="prqr_ClassesOfWarrantsOrRightsDomain_1" xlink:title="prqr_ClassesOfWarrantsOrRightsDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis_1" xlink:to="prqr_ClassesOfWarrantsOrRightsDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:prqr_IfrsClassesOfWarrantsOrRightsAxisAxis_1 To prqr_ClassesOfWarrantsOrRightsDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis" xlink:label="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis_1" xlink:title="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" xlink:label="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain_1" xlink:title="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis_1" xlink:to="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis_1 To ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain_1" order="1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_LiabilitiesArisingFromFinancingActivitiesAxis" xlink:label="ifrs-full_LiabilitiesArisingFromFinancingActivitiesAxis_1" xlink:title="ifrs-full_LiabilitiesArisingFromFinancingActivitiesAxis_1" />
    <link:loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_LiabilitiesArisingFromFinancingActivitiesDomain" xlink:label="ifrs-full_LiabilitiesArisingFromFinancingActivitiesDomain_1" xlink:title="ifrs-full_LiabilitiesArisingFromFinancingActivitiesDomain_1" />
    <link:definitionArc xlink:type="arc" xlink:from="ifrs-full_LiabilitiesArisingFromFinancingActivitiesAxis_1" xlink:to="ifrs-full_LiabilitiesArisingFromFinancingActivitiesDomain_1" use="optional" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:title="definition:ifrs-full_LiabilitiesArisingFromFinancingActivitiesAxis_1 To ifrs-full_LiabilitiesArisingFromFinancingActivitiesDomain_1" order="1" />
  </link:definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/StatementCondensedConsolidatedStatementOfChangesInEquity" xlink:type="extended" xlink:title="00300 - Statement - Condensed Consolidated Statement of Changes in Equity" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_StatementOfChangesInEquityTable" xlink:label="ifrs-full_StatementOfChangesInEquityTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ComponentsOfEquityAxis" xlink:label="ifrs-full_ComponentsOfEquityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_StatementOfChangesInEquityTable" xlink:to="ifrs-full_ComponentsOfEquityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ComponentsOfEquityDomain" xlink:label="ifrs-full_ComponentsOfEquityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_ComponentsOfEquityAxis" xlink:to="ifrs-full_ComponentsOfEquityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_IssuedCapitalMember" xlink:label="ifrs-full_IssuedCapitalMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_ComponentsOfEquityDomain" xlink:to="ifrs-full_IssuedCapitalMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SharePremiumMember" xlink:label="ifrs-full_SharePremiumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_ComponentsOfEquityDomain" xlink:to="ifrs-full_SharePremiumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ReserveOfSharebasedPaymentsMember" xlink:label="ifrs-full_ReserveOfSharebasedPaymentsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_ComponentsOfEquityDomain" xlink:to="ifrs-full_ReserveOfSharebasedPaymentsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ReserveOfExchangeDifferencesOnTranslationMember" xlink:label="ifrs-full_ReserveOfExchangeDifferencesOnTranslationMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_ComponentsOfEquityDomain" xlink:to="ifrs-full_ReserveOfExchangeDifferencesOnTranslationMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RetainedEarningsMember" xlink:label="ifrs-full_RetainedEarningsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_ComponentsOfEquityDomain" xlink:to="ifrs-full_RetainedEarningsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_StatementOfChangesInEquityLineItems" xlink:label="ifrs-full_StatementOfChangesInEquityLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_StatementOfChangesInEquityTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_Equity" xlink:label="ifrs-full_Equity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_Equity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NumberOfSharesOutstanding" xlink:label="ifrs-full_NumberOfSharesOutstanding" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_NumberOfSharesOutstanding" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProfitLoss" xlink:label="ifrs-full_ProfitLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_ProfitLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_OtherComprehensiveIncome" xlink:label="ifrs-full_OtherComprehensiveIncome" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_OtherComprehensiveIncome" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_IncreaseDecreaseThroughSharebasedPaymentTransactions" xlink:label="ifrs-full_IncreaseDecreaseThroughSharebasedPaymentTransactions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_IncreaseDecreaseThroughSharebasedPaymentTransactions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_IssueOfEquity" xlink:label="ifrs-full_IssueOfEquity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_IssueOfEquity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" xlink:label="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_TreasurySharesTransferred" xlink:label="prqr_TreasurySharesTransferred" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="prqr_TreasurySharesTransferred" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_SharesOptionsLapsed" xlink:label="prqr_SharesOptionsLapsed" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="prqr_SharesOptionsLapsed" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_IncreaseDecreaseThroughExerciseOfOptions" xlink:label="ifrs-full_IncreaseDecreaseThroughExerciseOfOptions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="ifrs-full_IncreaseDecreaseThroughExerciseOfOptions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="prqr_IfrsStockIssuedDuringPeriodSharesStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="ifrs-full_StatementOfChangesInEquityLineItems" xlink:to="prqr_IfrsStockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureGeneralInformationDetails" xlink:type="extended" xlink:title="40101 - Disclosure - General Information (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesTable" xlink:label="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInSubsidiariesAxis" xlink:label="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesTable" xlink:to="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:label="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesAxis" xlink:to="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsHoldingB.v.Member" xlink:label="prqr_ProqrTherapeuticsHoldingB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsHoldingB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIB.v.Member" xlink:label="prqr_ProqrTherapeuticsIB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIiB.v.Member" xlink:label="prqr_ProqrTherapeuticsIiB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIiB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIiiB.v.Member" xlink:label="prqr_ProqrTherapeuticsIiiB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIiiB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIvB.v.Member" xlink:label="prqr_ProqrTherapeuticsIvB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIvB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsVB.v.Member" xlink:label="prqr_ProqrTherapeuticsVB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsVB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsViB.v.Member" xlink:label="prqr_ProqrTherapeuticsViB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsViB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsViiB.v.Member" xlink:label="prqr_ProqrTherapeuticsViiB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsViiB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsViiiB.v.Member" xlink:label="prqr_ProqrTherapeuticsViiiB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsViiiB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIxB.v.Member" xlink:label="prqr_ProqrTherapeuticsIxB.v.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIxB.v.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProqrTherapeuticsIInc.Member" xlink:label="prqr_ProqrTherapeuticsIInc.Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="ifrs-full_SignificantInvestmentsInSubsidiariesDomain" xlink:to="prqr_ProqrTherapeuticsIInc.Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesLineItems" xlink:label="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesLineItems" xlink:to="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProportionOfOwnershipInterestInSubsidiary" xlink:label="ifrs-full_ProportionOfOwnershipInterestInSubsidiary" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfSignificantInvestmentsInSubsidiariesLineItems" xlink:to="ifrs-full_ProportionOfOwnershipInterestInSubsidiary" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureCriticalAccountingEstimatesAndJudgementsDetails" xlink:type="extended" xlink:title="40401 - Disclosure - Critical Accounting Estimates and Judgements (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:to="ifrs-full_CounterpartiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_EliLillyAndCompanyMember" xlink:label="prqr_EliLillyAndCompanyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_CounterpartiesDomain" xlink:to="prqr_EliLillyAndCompanyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DeferredIncomeIncludingContractLiabilities" xlink:label="ifrs-full_DeferredIncomeIncludingContractLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DeferredIncomeIncludingContractLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CashAndCashEquivalents" xlink:label="ifrs-full_CashAndCashEquivalents" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_CashAndCashEquivalents" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosurePropertyPlantAndEquipmentDetails" xlink:type="extended" xlink:title="40701 - Disclosure - Property, Plant and Equipment (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfPropertyPlantAndEquipmentTable" xlink:label="ifrs-full_DisclosureOfPropertyPlantAndEquipmentTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfPropertyPlantAndEquipmentTable" xlink:to="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:to="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_LeaseholdImprovementsMember" xlink:label="ifrs-full_LeaseholdImprovementsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:to="ifrs-full_LeaseholdImprovementsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfPropertyPlantAndEquipmentLineItems" xlink:label="ifrs-full_DisclosureOfPropertyPlantAndEquipmentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfPropertyPlantAndEquipmentLineItems" xlink:to="ifrs-full_DisclosureOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RightofuseAssets" xlink:label="ifrs-full_RightofuseAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfPropertyPlantAndEquipmentLineItems" xlink:to="ifrs-full_RightofuseAssets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureBorrowingsDetails" xlink:type="extended" xlink:title="40901 - Disclosure - Borrowings (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsTable" xlink:label="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_BorrowingsByNameAxis" xlink:label="ifrs-full_BorrowingsByNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsTable" xlink:to="ifrs-full_BorrowingsByNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_BorrowingsByNameDomain" xlink:label="ifrs-full_BorrowingsByNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_BorrowingsByNameAxis" xlink:to="ifrs-full_BorrowingsByNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_InnovationCreditMember" xlink:label="prqr_InnovationCreditMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_BorrowingsByNameDomain" xlink:to="prqr_InnovationCreditMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsClassesOfWarrantsOrRightsAxisAxis" xlink:label="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsTable" xlink:to="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ClassesOfWarrantsOrRightsDomain" xlink:label="prqr_ClassesOfWarrantsOrRightsDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="prqr_IfrsClassesOfWarrantsOrRightsAxisAxis" xlink:to="prqr_ClassesOfWarrantsOrRightsDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_PontifaxAndKreosWarrantsMember" xlink:label="prqr_PontifaxAndKreosWarrantsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="prqr_ClassesOfWarrantsOrRightsDomain" xlink:to="prqr_PontifaxAndKreosWarrantsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:label="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_Borrowings" xlink:label="ifrs-full_Borrowings" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_Borrowings" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InterestPayable" xlink:label="ifrs-full_InterestPayable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_InterestPayable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CurrentBorrowingsAndCurrentPortionOfNoncurrentBorrowings" xlink:label="ifrs-full_CurrentBorrowingsAndCurrentPortionOfNoncurrentBorrowings" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_CurrentBorrowingsAndCurrentPortionOfNoncurrentBorrowings" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NotionalAmount" xlink:label="ifrs-full_NotionalAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_NotionalAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_RepaymentsOfBorrowingsClassifiedAsFinancingActivities" xlink:label="ifrs-full_RepaymentsOfBorrowingsClassifiedAsFinancingActivities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="ifrs-full_RepaymentsOfBorrowingsClassifiedAsFinancingActivities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_CurrentBorrowingsExcludingAccruedInterest" xlink:label="prqr_CurrentBorrowingsExcludingAccruedInterest" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="prqr_CurrentBorrowingsExcludingAccruedInterest" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ConditionalWaiverOfDebtTotal" xlink:label="prqr_ConditionalWaiverOfDebtTotal" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="prqr_ConditionalWaiverOfDebtTotal" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_WarrantsEconomicLifetime" xlink:label="prqr_WarrantsEconomicLifetime" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_DisclosureOfDetailedInformationAboutBorrowingsLineItems" xlink:to="prqr_WarrantsEconomicLifetime" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureLeaseLiabilitiesDetails" xlink:type="extended" xlink:title="41001 - Disclosure - Lease Liabilities (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsTable" xlink:label="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsTable" xlink:to="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:label="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_ClassesOfPropertyPlantAndEquipmentAxis" xlink:to="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_OfficeAndLaboratoryFacilityMember" xlink:label="prqr_OfficeAndLaboratoryFacilityMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_ClassesOfPropertyPlantAndEquipmentDomain" xlink:to="prqr_OfficeAndLaboratoryFacilityMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_GeographicalAreasAxis" xlink:label="ifrs-full_GeographicalAreasAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsTable" xlink:to="ifrs-full_GeographicalAreasAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_GeographicalAreasDomain" xlink:label="ifrs-full_GeographicalAreasDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_GeographicalAreasAxis" xlink:to="ifrs-full_GeographicalAreasDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_LeidenNetherlandsMember" xlink:label="prqr_LeidenNetherlandsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_GeographicalAreasDomain" xlink:to="prqr_LeidenNetherlandsMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:label="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:to="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_AreaOfPropertyUnderLease" xlink:label="prqr_AreaOfPropertyUnderLease" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:to="prqr_AreaOfPropertyUnderLease" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_LeaseTerm" xlink:label="prqr_LeaseTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:to="prqr_LeaseTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_LeaseExtensionTerm" xlink:label="prqr_LeaseExtensionTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:to="prqr_LeaseExtensionTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_LeaseCurrentExtensionPeriodUnderOption" xlink:label="prqr_LeaseCurrentExtensionPeriodUnderOption" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_DisclosureOfQuantitativeInformationAboutRightofuseAssetsLineItems" xlink:to="prqr_LeaseCurrentExtensionPeriodUnderOption" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureDeferredIncomeDetails" xlink:type="extended" xlink:title="41101 - Disclosure - Deferred Income (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:to="ifrs-full_CounterpartiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_EliLillyAndCompanyMember" xlink:label="prqr_EliLillyAndCompanyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_CounterpartiesDomain" xlink:to="prqr_EliLillyAndCompanyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CurrentDeferredIncomeIncludingCurrentContractLiabilities" xlink:label="ifrs-full_CurrentDeferredIncomeIncludingCurrentContractLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_CurrentDeferredIncomeIncludingCurrentContractLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NoncurrentDeferredIncomeIncludingNoncurrentContractLiabilities" xlink:label="ifrs-full_NoncurrentDeferredIncomeIncludingNoncurrentContractLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_NoncurrentDeferredIncomeIncludingNoncurrentContractLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DeferredIncomeIncludingContractLiabilities" xlink:label="ifrs-full_DeferredIncomeIncludingContractLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DeferredIncomeIncludingContractLiabilities" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureShareholdersEquityShareCapitalDetails" xlink:type="extended" xlink:title="41201 - Disclosure - Shareholders' Equity - Share capital (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfClassesOfShareCapitalTable" xlink:label="ifrs-full_DisclosureOfClassesOfShareCapitalTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfShareCapitalAxis" xlink:label="ifrs-full_ClassesOfShareCapitalAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalTable" xlink:to="ifrs-full_ClassesOfShareCapitalAxis" priority="2" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ClassesOfShareCapitalDomain" xlink:label="ifrs-full_ClassesOfShareCapitalDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_ClassesOfShareCapitalAxis" xlink:to="ifrs-full_ClassesOfShareCapitalDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_OrdinarySharesMember" xlink:label="ifrs-full_OrdinarySharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_ClassesOfShareCapitalDomain" xlink:to="ifrs-full_OrdinarySharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_PreferenceSharesMember" xlink:label="ifrs-full_PreferenceSharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_ClassesOfShareCapitalDomain" xlink:to="ifrs-full_PreferenceSharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalTable" xlink:to="ifrs-full_CounterpartiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_EliLillyAndCompanyMember" xlink:label="prqr_EliLillyAndCompanyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_CounterpartiesDomain" xlink:to="prqr_EliLillyAndCompanyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:label="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_DisclosureOfClassesOfShareCapitalTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_AuthorizedShareCapitalValue" xlink:label="prqr_AuthorizedShareCapitalValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_AuthorizedShareCapitalValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NumberOfSharesAuthorised" xlink:label="ifrs-full_NumberOfSharesAuthorised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_NumberOfSharesAuthorised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NumberOfSharesIssued" xlink:label="ifrs-full_NumberOfSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_NumberOfSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NumberOfSharesIssuedAndFullyPaid" xlink:label="ifrs-full_NumberOfSharesIssuedAndFullyPaid" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_NumberOfSharesIssuedAndFullyPaid" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_SharesInEntityHeldByEntityOrByItsSubsidiariesOrAssociates" xlink:label="ifrs-full_SharesInEntityHeldByEntityOrByItsSubsidiariesOrAssociates" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_SharesInEntityHeldByEntityOrByItsSubsidiariesOrAssociates" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ParValuePerShare" xlink:label="ifrs-full_ParValuePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_ParValuePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" xlink:label="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_IssueOfEquity" xlink:label="ifrs-full_IssueOfEquity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_IssueOfEquity" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureShareholdersEquitySaleOfSharesDetails" xlink:type="extended" xlink:title="41202 - Disclosure - Shareholders' Equity - Sale of shares (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfClassesOfShareCapitalTable" xlink:label="ifrs-full_DisclosureOfClassesOfShareCapitalTable" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockAxis" xlink:label="prqr_IfrsSaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalTable" xlink:to="prqr_IfrsSaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockDomain" xlink:label="prqr_IfrsSaleOfStockDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="prqr_IfrsSaleOfStockAxis" xlink:to="prqr_IfrsSaleOfStockDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ShelfRegistrationStatement2024Member" xlink:label="prqr_ShelfRegistrationStatement2024Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="prqr_IfrsSaleOfStockDomain" xlink:to="prqr_ShelfRegistrationStatement2024Member" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_AtMarketOfferingsWithCantorFitzgeraldAndCoUnderShelfRegistrationStatementMember" xlink:label="prqr_AtMarketOfferingsWithCantorFitzgeraldAndCoUnderShelfRegistrationStatementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="prqr_ShelfRegistrationStatement2024Member" xlink:to="prqr_AtMarketOfferingsWithCantorFitzgeraldAndCoUnderShelfRegistrationStatementMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_October2024UnderwrittenPublicOfferingMember" xlink:label="prqr_October2024UnderwrittenPublicOfferingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="prqr_ShelfRegistrationStatement2024Member" xlink:to="prqr_October2024UnderwrittenPublicOfferingMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_UnderwritersOptionInConnectionToOctober2024UnderwrittenPublicOfferingMember" xlink:label="prqr_UnderwritersOptionInConnectionToOctober2024UnderwrittenPublicOfferingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="prqr_October2024UnderwrittenPublicOfferingMember" xlink:to="prqr_UnderwritersOptionInConnectionToOctober2024UnderwrittenPublicOfferingMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_SharePurchaseAgreementWithEliLillyAndCompanyMember" xlink:label="prqr_SharePurchaseAgreementWithEliLillyAndCompanyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="prqr_ShelfRegistrationStatement2024Member" xlink:to="prqr_SharePurchaseAgreementWithEliLillyAndCompanyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_SharePurchaseAgreementWithGinkgoBioworksMember" xlink:label="prqr_SharePurchaseAgreementWithGinkgoBioworksMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="prqr_ShelfRegistrationStatement2024Member" xlink:to="prqr_SharePurchaseAgreementWithGinkgoBioworksMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_June2026UnderwrittenPublicOfferingMember" xlink:label="prqr_June2026UnderwrittenPublicOfferingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="prqr_ShelfRegistrationStatement2024Member" xlink:to="prqr_June2026UnderwrittenPublicOfferingMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:label="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_DisclosureOfClassesOfShareCapitalTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MaximumOfferingPriceInShelfRegistrationStatement" xlink:label="prqr_MaximumOfferingPriceInShelfRegistrationStatement" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_MaximumOfferingPriceInShelfRegistrationStatement" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_PercentageOfCommissionPayableForGrossProceedsFromSalesAgreement" xlink:label="prqr_PercentageOfCommissionPayableForGrossProceedsFromSalesAgreement" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_PercentageOfCommissionPayableForGrossProceedsFromSalesAgreement" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" xlink:label="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSaleOfStockPricePerShare" xlink:label="prqr_IfrsSaleOfStockPricePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_IfrsSaleOfStockPricePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_DurationOfUnderwritersOption" xlink:label="prqr_DurationOfUnderwritersOption" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_DurationOfUnderwritersOption" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_NumberOfSharesCallableForOptionsIssued" xlink:label="prqr_NumberOfSharesCallableForOptionsIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_NumberOfSharesCallableForOptionsIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProceedsFromIssuingShares" xlink:label="ifrs-full_ProceedsFromIssuingShares" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_ProceedsFromIssuingShares" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_PaymentsForShareIssueCosts" xlink:label="ifrs-full_PaymentsForShareIssueCosts" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="ifrs-full_PaymentsForShareIssueCosts" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_NetProceedsFromIssuingShares" xlink:label="prqr_NetProceedsFromIssuingShares" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_NetProceedsFromIssuingShares" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_PurchasePriceCapSharePurchaseAgreement" xlink:label="prqr_PurchasePriceCapSharePurchaseAgreement" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_PurchasePriceCapSharePurchaseAgreement" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ReferencePricePerShare" xlink:label="prqr_ReferencePricePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_ReferencePricePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ContractualCreditReceivedAsConsiderationForSharesIssued" xlink:label="prqr_ContractualCreditReceivedAsConsiderationForSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_ContractualCreditReceivedAsConsiderationForSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ContractualTermOverWhichCreditCanBeUtilized" xlink:label="prqr_ContractualTermOverWhichCreditCanBeUtilized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="ifrs-full_DisclosureOfClassesOfShareCapitalLineItems" xlink:to="prqr_ContractualTermOverWhichCreditCanBeUtilized" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureShareholdersEquityShareOptionsDetails" xlink:type="extended" xlink:title="41203 - Disclosure - Shareholders' Equity - Share options (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementTable" xlink:label="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" xlink:label="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementTable" xlink:to="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" xlink:label="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionAxis" xlink:to="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsSellingGeneralAndAdministrativeExpensesMember" xlink:label="prqr_IfrsSellingGeneralAndAdministrativeExpensesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" xlink:to="prqr_IfrsSellingGeneralAndAdministrativeExpensesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsResearchAndDevelopmentExpenseMember" xlink:label="prqr_IfrsResearchAndDevelopmentExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_AttributionOfExpensesByNatureToTheirFunctionDomain" xlink:to="prqr_IfrsResearchAndDevelopmentExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementLineItems" xlink:label="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementLineItems" xlink:to="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ExpenseFromEquitysettledSharebasedPaymentTransactionsInWhichGoodsOrServicesReceivedDidNotQualifyForRecognitionAsAssets" xlink:label="ifrs-full_ExpenseFromEquitysettledSharebasedPaymentTransactionsInWhichGoodsOrServicesReceivedDidNotQualifyForRecognitionAsAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfTermsAndConditionsOfSharebasedPaymentArrangementLineItems" xlink:to="ifrs-full_ExpenseFromEquitysettledSharebasedPaymentTransactionsInWhichGoodsOrServicesReceivedDidNotQualifyForRecognitionAsAssets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureRevenueDetails" xlink:type="extended" xlink:title="41301 - Disclosure - Revenue (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:to="ifrs-full_CounterpartiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_EliLillyAndCompanyMember" xlink:label="prqr_EliLillyAndCompanyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_CounterpartiesDomain" xlink:to="prqr_EliLillyAndCompanyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProductsAndServicesAxis" xlink:label="ifrs-full_ProductsAndServicesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" xlink:to="ifrs-full_ProductsAndServicesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProductsAndServicesDomain" xlink:label="ifrs-full_ProductsAndServicesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_ProductsAndServicesAxis" xlink:to="ifrs-full_ProductsAndServicesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MilestonePaymentsMember" xlink:label="prqr_MilestonePaymentsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_ProductsAndServicesDomain" xlink:to="prqr_MilestonePaymentsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:label="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_DeferredIncomeIncludingContractLiabilities" xlink:label="ifrs-full_DeferredIncomeIncludingContractLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_DeferredIncomeIncludingContractLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" xlink:label="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="prqr_IfrsStockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_NumberOfSharesIssued" xlink:label="ifrs-full_NumberOfSharesIssued" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_NumberOfSharesIssued" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ProceedsFromIssuingShares" xlink:label="ifrs-full_ProceedsFromIssuingShares" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_ProceedsFromIssuingShares" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_DiscountOnIssueOfShares" xlink:label="prqr_DiscountOnIssueOfShares" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="prqr_DiscountOnIssueOfShares" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_AdditionalConsiderationToBeReceivedIfCounterpartyExercisesOptionToExpandPartnership" xlink:label="prqr_AdditionalConsiderationToBeReceivedIfCounterpartyExercisesOptionToExpandPartnership" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="prqr_AdditionalConsiderationToBeReceivedIfCounterpartyExercisesOptionToExpandPartnership" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_NumberOfPerformanceObligationsInCollaborationAgreement" xlink:label="prqr_NumberOfPerformanceObligationsInCollaborationAgreement" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="prqr_NumberOfPerformanceObligationsInCollaborationAgreement" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" xlink:label="ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ValueOfMilestonesReachedDuringPeriod" xlink:label="prqr_ValueOfMilestonesReachedDuringPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="ifrs-full_DisclosureOfDisaggregationOfRevenueFromContractsWithCustomersLineItems" xlink:to="prqr_ValueOfMilestonesReachedDuringPeriod" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureOtherIncomeNarrativeDetails" xlink:type="extended" xlink:title="41402 - Disclosure - Other Income - Narrative (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_OtherIncomeTable" xlink:label="prqr_OtherIncomeTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesAxis" xlink:label="ifrs-full_CounterpartiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="prqr_OtherIncomeTable" xlink:to="ifrs-full_CounterpartiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_CounterpartiesDomain" xlink:label="ifrs-full_CounterpartiesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_CounterpartiesAxis" xlink:to="ifrs-full_CounterpartiesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_RettSyndromeResearchTrustMember" xlink:label="prqr_RettSyndromeResearchTrustMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_CounterpartiesDomain" xlink:to="prqr_RettSyndromeResearchTrustMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_OtherIncomeLineItems" xlink:label="prqr_OtherIncomeLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="prqr_OtherIncomeLineItems" xlink:to="prqr_OtherIncomeTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_OtherIncome" xlink:label="ifrs-full_OtherIncome" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="prqr_OtherIncomeLineItems" xlink:to="ifrs-full_OtherIncome" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_IfrsRevenueFromGrants" xlink:label="prqr_IfrsRevenueFromGrants" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="prqr_OtherIncomeLineItems" xlink:to="prqr_IfrsRevenueFromGrants" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MaximumGrantReceivableForInitialPhaseOfProject" xlink:label="prqr_MaximumGrantReceivableForInitialPhaseOfProject" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="prqr_OtherIncomeLineItems" xlink:to="prqr_MaximumGrantReceivableForInitialPhaseOfProject" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_MaximumGrantReceivableForAdditionalPhaseOfProject" xlink:label="prqr_MaximumGrantReceivableForAdditionalPhaseOfProject" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="prqr_OtherIncomeLineItems" xlink:to="prqr_MaximumGrantReceivableForAdditionalPhaseOfProject" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" xlink:label="ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="prqr_OtherIncomeLineItems" xlink:to="ifrs-full_ReceiptsFromSalesOfGoodsAndRenderingOfServices" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.proqr.com/role/DisclosureInvestmentsInFinancialAssetsDetails" xlink:type="extended" xlink:title="41701 - Disclosure - Investments in Financial Assets (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateTable" xlink:label="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateTable" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis" xlink:label="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateTable" xlink:to="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" xlink:label="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="ifrs-full_InvestmentsInEquityInstrumentsMeasuredAtFairValueThroughOtherComprehensiveIncomeAxis" xlink:to="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_PhoenicisTherapeuticsIncMember" xlink:label="prqr_PhoenicisTherapeuticsIncMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" xlink:to="prqr_PhoenicisTherapeuticsIncMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_YarrowBiotechnologyMember" xlink:label="prqr_YarrowBiotechnologyMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="ifrs-full_InvestmentsInEquityInstrumentsDesignatedAtFairValueThroughOtherComprehensiveIncomeDomain" xlink:to="prqr_YarrowBiotechnologyMember" use="optional" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateLineItems" xlink:label="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateLineItems" xlink:to="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="prqr-20260630.xsd#prqr_ProportionOfOwnershipInterest" xlink:label="prqr_ProportionOfOwnershipInterest" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateLineItems" xlink:to="prqr_ProportionOfOwnershipInterest" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="https://xbrl.ifrs.org/taxonomy/2025-03-27/full_ifrs/full_ifrs-cor_2025-03-27.xsd#ifrs-full_FinancialAssetsAtFairValueThroughOtherComprehensiveIncome" xlink:label="ifrs-full_FinancialAssetsAtFairValueThroughOtherComprehensiveIncome" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="prqr_DisclosureOfInvestmentInFinancialAssetAndGainOnDisposalOfAssociateLineItems" xlink:to="ifrs-full_FinancialAssetsAtFairValueThroughOtherComprehensiveIncome" priority="1" use="optional" />
  </definitionLink>
</link:linkbase>
