The aim of the investigation was to assess the availability of ozone to induce DNA damage in individual cells when analyzing them using the Comet assay. Materials and Methods. Experimental studies were performed on whole blood leukocytes of white non-linear intact male rats (n=16) weighing 250±25 g. Two series of experiments were made to induce DNA damage in leukocytes. During the first series the samples were exposed to gamma-radiation, and during the second series the slides were treated with ozonized phosphate buffer saline. Further the cells were exposed to cytolysis followed by DNA denaturation, electrophoresis, neutralization, DNA being stained with a SYBR GREEN I. Comet visualization (fluorescent microscopy) and scoring were performed. Results. The new version of the Comet assay was developed. Ozone concentration, 900 μg/L, in ozone-oxygen mixture, and the exposure time for 10 min on the cells on a microscope slides were found to be optimal for detection of DNA damage and its analysis. In addition, ozone application enables to minimize the drawbacks and limitations of gamma-radiation source.