Decay of an Auger-electron-emitting radioisotope can knock out a targeted gene by producing DNA strand breaks within its sequence. For delivery of Auger emitters to genomic targets we used triplex-forming oligonucleotides (TFOs) that bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. We named this approach antigene radiotherapy. In our previous studies, we demonstrated that ¹²⁵I-labeled TFOs targeted against the human MDR1 gene produced sequence-specific double strand breaks (DSBs) within this gene in live cultured cells. We also found that conjugation of TFO with nuclear localization signal peptide significantly increased the efficiency of targeting. To screen the wide variety of possible TFO modifications a sensitive and robust assay of DNA damage produced by such ¹²⁵I-TFOs would be highly desirable. Recently we showed a direct correspondence between the number of decays of ¹²⁵I incorporated into DNA as ¹²⁵I-UdR and the number of histone -H2AX foci per cell revealed by staining with -H2AX antibodies. The technique is 100-fold more sensitive than other DSB-detection methods, thus it is possible to detect as few as an average of 0.5 DSBs per cell in a population of cultured cells. Here we applied this method to evaluate the intracellular DNA damage produced by two ¹²⁵I-TFOs, the first targeted to the single-copy HPRT gene (¹²⁵I-TFO-HPRT) and second to a multicopy repeated sequence (GA)_n that occurs almost 7000 times in the human genome (¹²⁵I-TFO-GA). DNA damage produced by ¹²⁵I-TFO was assessed by staining the cells with -H2AX antibody followed by either direct counting -H2AX foci or by measuring the -H2AX signal using flow cytometry. Both methods produced quantitatively close results; ¹²⁵I-TFO-GA with multiple nuclear targets produced on average 1.93 times more -H2AX foci per cell and generated 1.96 times increase in -H2AX antibody staining signal than ¹²⁵I-TFO-HPRT with a single target. The -H2AX-based assay requires considerably less time and effort than the direct measurement of DSB by Southern hybridization applied previously. Therefore, we believe that -H2AX-based measurement of DNA damage could be useful for evaluation and cellular DNA accessibility by ¹²⁵I-labeled DNA targeting agents.