In vivo validation of models of DNA damage repair will enable their use for optimizing clinical radiotherapy. In this study, a theoretical assessment was made of DNA double-strand break (DSB) induction in normal breast tissue after intraoperative radiation therapy (IORT), which is now an accepted form of adjuvant radiotherapy for selected patients with early breast cancer. DSB rates and relative biological effectiveness (RBE) were calculated as a function of dose, radiation quality and dose rate, each varying based on the applicator size used during IORT. The spectra of primary electrons in breast tissue adjacent to each applicator were calculated using measured X-ray spectra and Monte Carlo methods, and were used to inform a Monte Carlo damage simulation code. In the absence of repair, asymptotic RBE values (relative to ⁶⁰Co) were approximately 1.5. Beam-quality changes led to only minor variations in RBE among applicators, though differences in dose rate and overall dose delivery time led to larger variations and a rapid decrease in RBE. An experimental assessment of DSB induction was performed ex vivo using pre- and postirradiation tissue samples from patients receiving breast intraoperative radiation therapy. Relative DSB rates were assessed via γ-H2AX immunohistochemistry using proportional staining. Maximum-likelihood parameter estimation yielded a DSB repair halftime of 25.9 min (95% CI, 21.5–30.4 min), although the resulting model was not statistically distinguishable from one where there was no change in DSB yield among patients. Although the model yielded an in vivo repair halftime of the order of previous estimates for in vitro repair halftimes, we cannot conclude that it is valid in this context. This study highlights some of the uncertainties inherent in population analysis of ex vivo samples, and of the quantitative limitations of immunohistochemistry for assessment of DSB repair.