Genotoxicity tests are an important part of drug safety evaluation and play a crucial role in ensuring drug safety. In recent years, as domestic and international regulatory agencies actively promote animal alternative testing, the importance of in vitro genotoxicity tests has grown significantly. It is urgent to strengthen the standardization construction, optimization and promotion of relevant standards. The methods of in vitro genotoxicity tests cover bacterial-based genotoxicity tests and mammalian cell-based genotoxicity tests. Due to being closer to the human environment, the results of mammalian cell-based genotoxicity tests will be more trusted by people. The Mouse Lymphoma Cell TK Gene Mutation Assay (MLA) is favored by evaluators due to its higher similarity to the human environment and the ability to simultaneously identify gene mutations and chromosomal damage. It is increasingly applied in various fields. Based on the latest guidance principles of regulatory agencies regarding genotoxicity tests at home and abroad, through literature review, this article summarizes the test principles, applications and limitations of MLA, to guide the application of MLA and provide a reference for its applicable scope, helping to enhance the scientificity and accuracy of genotoxicity evaluation applications.