Experimental drugs need to be screened for safety within time constraints. Hepatotoxicity is one concerning contributor to the failure of investigational new drugs and a major rationale for postmarketing withdrawal decisions. Ethical considerations in preclinical research force the requirement for highly predictive in vitro assays using human tissue which retains functionality reflective of primary tissue. Here, the proteome of cells commonly used to assess preclinical hepatotoxicity was compared. Primary human hepatocytes (PHHs), hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells, HepG2 cell monolayers and HepG2 cell 3D spheroids were cultured and collected as whole cell lysates. Over 6000 proteins were identified and quantified in terms of relative abundance in replicate proteomic experiments using isobaric tagging methods. Comparison of these quantitative data provides biological insight into the feasibility of using HLCs, HepG2 monolayers, and HepG2 3D spheroids for hepatotoxicity testing. Collectively these data reveal how HLCs differentiated for 35 days and HepG2 cells proteomes differ from one another and that of PHHs. HepG2 cells possess a strong cancer cell signature and do not adequately express key metabolic proteins which mark the hepatic phenotype, this was not substantially altered by culturing as 3D spheroids. These data suggest that while no single hepatic model reflects the diverse array of outcomes required to mimic the in vivo liver functions, that HLCs are the most suitable investigational avenue for replacing PHHs in vitro.