Most anticancer treatments such as chemo- and radiotherapy induce DNA damage and apoptosis in normal cells. The aim of this study was to assess the induction of single and double DNA strand breaks (ssb and dsb, respectively) and apoptosis in normal human lymphocytes and rat thymocytes subjected to the action of H2O2, bleomycin and ionizing radiation. Normal human peripheral thymocytes and young rat thymocytes were subjected to the following treatments: a) H2O2; b) bleomycin, and c) gamma-radiation, all with various doses. DNA strand breaks were studied with the alkaline and neutral comet assay for detection of ssb and dsb. Apoptosis was quantified morphologically and with DNA agarose gel electrophoresis. After H2O2 treatment, a dose-dependent increase of ssb was observed. Bleomycin treatment produced a moderate increase of ssb at lower concentrations and a striking increase of dsb at higher concentrations that coincided with the presence of apoptosis and DNA ladders. Gamma radiation initially induced the formation of ssb, and after three hours an increase of dsb in a dose-dependent manner. Apoptosis and DNA laddering appeared only 3 hours post-irradiation. The biomonitoring of DNA damage inflicted by antineoplastic agents can be easily performed with the comet assay and could be useful to monitor and modulate chemo- and radiotherapeutic regimes in cancer patients.