Scientifically credible and valid biological systems are essential in pharmaceutical research and development. Standardizing in vitro preclinical hepatotoxicity is confounded by the diversity of origin of cells and the ability to retain hepatocellular functions. Key determinants of valid hepatotoxicity models are resemblance to primary human hepatocytes (PHHs), adaptability to high-throughput screening and biological applicability. Numerous in vitro models, including immortalized cell lines and hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs), attempt to reflect features of PHH. Additional influencing factors are the mechanical and geometric environment which dictate functionality and suggest a role for spatial organization as a requirement for mimicking PHH. As there is poor correlation between the cellular genome and proteome, assessing the hepatic phenotypes using proteomics is essential to capture functional cellular responses. The aim of this research was to determine proteomic differences between PHHs and differentially cultured and sourced human hepatocyte-derived cell lines or differentiated HLCs. Additionally, hepatocyte models were used to generate non-specific, proteome-wide information associated with exposure to selected known hepatotoxins to identify potential proteomic signatures of hepatotoxicity.