Background: Standardizing in vitro pre-clinical hepatotoxicity is confounded by diverse cellular origins which often lack representative hepatocellular function. Dedifferentiation and loss of cellular polarity are inherent limitations introduced by long term cell culture. HepG2 cells, with a stable phenotype in early passage, have been extensively used in hepatotoxicity screening despite an apparent poor metabolic competence and "foetal-like" hepatic phenotype. Three dimensional (3D) culture techniques may serve to better preserve the cellular phenotype. One strategy to assess the differences in hepatic phenotypes is assessing the proteome. The aim of this research was to determine abundance changes in the hepatic proteome of HepG2 cells when cultured in "hanging drop" 3D cultures. Methods: Replicates of HepG2 cells (as monolayers and spheroids) were collected as whole cell lysates. Fifty micrograms of sample protein was reduced, alkylated, precipitated, digested with trypsin and labelled using isobaric tagging. Samples were analysed using a Dionex Ultimate 3000 RSLCnano LC system coupled to a Thermo Scientific Fusion Orbitrap Mass Spectrometer. Peak lists were searched against a UniProtKB/Swiss-Prot human database using SearchGUI version 2.3.1 with X!Tandem, MS-GF+ and Comet search engines. Targeted acquisitions were carried out using Skyline software. Results: Once digitally filtered, 4701, 5030 and 4932 proteins were identified and quantified across replicates 1 to 3 with a protein overlap of 3760. While distinct hepatic marker profiles varied between samples, several notable proteins were upregulated across culture models. Among these were hallmark hepatic proteins albumin and alpha-fetoprotein which showed 3.6 and 6.1-fold increases respectively. Canalicular specific marker proteins such as dipeptidyl peptidase 4 were identified with a 2-fold up-regulated in spheroid cultures. Hepatic structural markers such as hepatocyte cell adhesion molecule, collagen, laminin and fibronectin were also up-regulated by more than 2-fold. Several phase I metabolizing enzymes and membrane transporter proteins showed variable up-regulation. Conclusion: Hallmark hepatic proteins were largely up-regulated, demonstrating alterations in the hepatic phenotype, when cultured in 3D spheroid free from synthetic surfaces. Quantitation of large protein cohorts demonstrated the ability of 3D spheroids to induce changes in the HepG2 cell proteome, these changes could be advantageous in promoting maintenance of a stable hepatic phenotype.