Human hepatic cell lines have been widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins including drug-metabolizing enzymes (DMEs) in the cell lines can differ significantly from that of human livers. In the present study, we first conducted an untargeted proteomics of the microsomes of the cell lines HepG2, Hep3B, and Huh7 in comparison with human livers using a SWATH method. Furthermore, a targeted proteomic approach, named high-resolution multiple reaction monitoring (MRM-HR), was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines.