Abstract: Hepatotoxicity of drugs is one of the important contents of drug safety evaluation. The accurate evaluation of potential hepatotoxicity of drugs and their metabolites in the early research and development is of great significance to improve the success rate of new drugs. Metabolomics technology combined with in vitro cell model can directly reflect the toxic effect and toxic mechanism of drugs by the change of cell metabolic phenotype, and can improve the prediction of preclinical drug hepatotoxicity, which has great application value and development potential in drug hepatotoxicity screening. This paper reviewed the current cell models and culture methods in hepatotoxicity research, introduced the advantages of 3D cell models in vitro studies, and summarized mass spectrometry-based metabolomics strategy in cell model and its application in the evaluation of drug hepatotoxicity. Among them, the spatially resolved metabolomics method based on mass spectrometry imaging technology has unique advantages in the study of cell models in vitro, which is expected to become a useful tool for the study of hepatotoxicity in vitro.