Hepatoma cell lines are frequently used as in vitro alternatives to primary human hepatocytes. Cell lines are characterized by their unlimited life span, stable phenotype, high availability, and easy handling. However, their major limitation is the lower expression of some metabolic activities compared with hepatocytes. HepG2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. HepG2 cells are nontumorigenic cells with high proliferation rates and an epithelial-like morphology that perform many differentiated hepatic functions. In this chapter, freezing, thawing, and subculturing procedures for HepG2 cells are described. We further provide protocols for evaluating lipid accumulation, glycogen storage, urea synthesis, and phase I and phase II drug metabolizing activities in HepG2 cells.