The in vitro micronucleus assay is a widely used method to assess the genotoxic potential of chemical compounds by detecting micronuclei in the cytoplasm of binucleated cells. This protocol describes the application of the micronucleus assay in Chinese hamster ovary (CHO-K1) cells, a well-established mammalian cell line. In this assay, cells are exposed to the test substance, followed by a determination of micronuclei frequency in binucleated cells, which serves as an indicator of chromosomal damage or mitotic disruption. The binucleated CHO-K1 cell model allows for the evaluation of both the clastogenic and aneugenic effects of test chemicals. The protocol includes detailed procedures for cell culture, treatment conditions, harvesting, and scoring of micronuclei. Additionally, the scoring criteria and statistical methods for data analysis are outlined to ensure consistent and reliable results. This assay serves as an essential tool in genotoxicity screening and risk assessment for chemicals in regulatory toxicology.