PurposeThis study aims to standardize the simulation procedure in measuring DNA double‐strand breaks (DSBs), by using advanced Monte Carlo toolkits, and newly introduced experimental methods for DNA DSB measurement.MethodsFor the experimental quantification of DNA DSB, an innovative DNA dosimeter was used to produce experimental data. GATE in combination with Geant4‐DNA toolkit were exploited to simulate the experimental environment. The PDB4DNA example of Geant4‐DNA was upgraded and investigated. Parameters of the simulation such energy threshold (ET) for a strand break and base pair threshold (BPT) for a DSB were evaluated, depending on the dose.ResultsSimulations resulted to minimum differentiation in comparison to experimental data for ET = 19 ± 1 eV and BPT = 10 bp, and high differentiation for ET<17.5 eV or ET>22.5 eV and BPT = 10 bp. There was also small differentiation for ET = 17.5 eV and BPT = 6 bp. Uncertainty has been kept lower than 3%.ConclusionsThis study includes first results on the quantification of DNA double‐strand breaks. The energy spectrum of a LINAC was simulated and used for the first time to irradiate DNA molecules. Simulation outcome was validated on experimental data that were produced by a prototype DNA dosimeter.