The comet assay (single-cell gel electrophoresis) is a widely used in vitro method for assessing genotoxicity, specifically DNA damage, within individual cells. This protocol outlines the procedure for evaluating genotoxicity in Chinese hamster ovary (CHO-K1) cells, a commonly used mammalian cell line in toxicology studies. The assay detects DNA strand breaks, alkali-labile sites, and cross-links by subjecting lysed cells to electrophoresis, followed by visualization and quantification of comet-like tail formation under a fluorescence microscope. The extent of DNA damage is determined by measuring parameters such as tail length, tail intensity, and tail moment. This protocol provides detailed steps for cell culture, treatment with genotoxic agents, preparation of slides, electrophoresis, and image analysis. The results from this assay can help identify potential genotoxic compounds, contributing to safety evaluation in pharmaceuticals, chemicals, and environmental agents.