Hepatocytes are known to express a large number of characteristic proteins. Transformed and cultured hepatocytes only partially maintain functional cell differentiation characteristics, which can be assessed by proteome profiling. Here, we applied 2D-PAGE analysis in addition to shotgun proteomics to assess the functional cell state of primary human hepatocytes (PHH), HepG2 and Hep3B cells. Out of a total of 1995 proteins identified in the cytoplasm of these cells, we filtered 107 proteins which are characteristic for hepatocytes. A total of 104 of those were identified in primary human hepatocytes, 20 in HepG2, and only 6 in Hep3B. Forty-six out of 72 proteins identified in the secretome of PHH, 55 out of 139 in HepG2, and only 24 out of 72 in Hep3B were plasma proteins characteristic for hepatocytes. Beside other biomarker candidates presently identified, 11 proteins of the HepG2 secretome have been described previously as biomarkers for hepatocellular carcinoma. Because of indications that epithelial to mesenchymal transition (EMT) may have occurred in the cultured hepatoma cells, we included the analysis of fibroblasts representative for mesenchymal cells. Hep3B, but not HepG2, secreted five proteins including follistatin-related protein 1 which are characteristic for mesenchymal cells and may be marker proteins for EMT. Our data demonstrate that HepG2 show more features characteristic for hepatocytes than Hep3B, while Hep3B express more mesenchymal proteins indicative for EMT. Proteome profiling thus proved to enable comprehensive assessment of functional cell states and cell differentiation states of cultured hepatocytes and enabled the identification of numerous biomarkers for hepatocellular carcinoma and EMT.