Despite the importance of hepatotoxicity testing in the development of new potential pharmaceuticals, standardized methods for preclinical in vitro hepatotoxicity is complicated by the perceived adequacy of approach, diversity of origin of cells, and the ability to retain a satisfactory hepatocellular phenotype. Additionally, the confidence with which cells mimic in vitro hepatocytes is dictated by the spatial dynamics of the cell culture microenvironment. This study sought to compare the proteome of conventional monolayer cultures of an immortalized hepatocyte cell line (HepG2) with more complex three-dimensional spheroid cultures to ascertain whether changes in culture technique better mimic the phenotype of hepatocytes and thereby improve responses to in vivo hepatotoxins. The proteome was assayed using isobaric tagging from six independent experiments, yielding relative quantitation of over 4600 proteins per multiplexed set. Approximately 34% of proteins present in all replicates differed between monolayer and 3D spheroid cultures. These data suggest that the cellular transition from an exponential to an equilibrium growth phase is inconsistent across biological replicates during spheroid formation which then variably alters the proteome from a stable phenotype in monolayers. Continuous exposure to hepatotoxins, did not implicate specific subsets of proteins in describing the associated mechanisms of toxicity of each drug. However, dynamic changes in HepG2 cells cultured as 3D spheroids were described. These data suggest that the duration of spheroid culture could be essential to reconcile the differences observed in the spheroid proteome to achieve reproducible proteomic transitions to a stable 3D spheroid phenotype.