Probiotics are live bacteria that show health advantages when given in sufficient quantities. They have gained a lot of interest because of their ability to improve gut health, boost immunity, and alter a number of physiological processes. However, differences in probiotic strains, doses, and host responses underline the need for standardized in vivo investigations to fully analyze their genotoxic profile and ensure long-term safety. According to the studies, most probiotic strains have no genotoxic effects and may even protect against DNA damage caused by environmental contaminants. This shows that probiotics may have antigenotoxic and antioxidant capabilities, increasing their safety and therapeutic potential. The comet assay (single-cell gel electrophoresis) is a sensitive method for detecting DNA strand breaks and oxidative damage at the cellular level, offering insights into the potential genotoxicity. This chapter explains the methodology to analyze the genotoxic potential of probiotics using comet assay.