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Aim: Comparative proteomics provide a powerful approach in screening for alter-ations in protein levels and post-translational modifications that are associated with tumors. In the present study, we aimed to identify candidate biomarkers to distinguish osteosarcoma (OS) cells from normal osteoblastic cells.Methods: We employed 30S cell lines (U2OS, IOR/OS9, and SaOS-2), and used the SV40-immor-talized normal osteoblastic cell line (hFOB 1.19) as the control. The differential protein levels in OS and osteoblastic cells were identified using 2-D gel electro-phoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses. Two proteins of interest, the levels of which were significantly increased in OS cells, were further characterized by Weste blot analyses.Results: Twenty-six proteins were identified, the expression level of which was either significantly increased or decreased in the OS cells as compared to the control cells. The expression level of the activator of 90 kDa shock protein ATPase homolog 1 (AHA1), was enhanced 12.4-, 24.1-, and 23.8-fold in SaOS-2,IOR/OS9, and U2OS cells, respectively, and the level of the stomatin-like protein 2 (SLP-2) was increased by 10.4- and 7.8-fold in IOR/OS9 and U2OS cells, respectively,as compared to normal osteoblastic cells. Those observations were confirmed by Weste blot analyses.Conclusion: A differential proteomic analysis was suc-cessfully used to identify AHA1 and SLP-2 that were significantly overproduced in OS cells as compared to normal osteoblastic cells, suggesting that those pro-teins among others may be effective biomarker candidates for the identification of OS cells.