Abstract: Objective To establish and optimize two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) for comparative analysis of the protein expression profiles in mouse fibroblast NIH3T3 cells before and after 12-O-tetradecanoy- lphorbol-13-acetate (TPA) treatment using image analysis software, so as to prepare for more intensive study in the identifi- cation of the proteins that mediate the biological effect of TPA. Methods The total cellular protein extracted from NIH3T3 cells with or without TPA treatment underwent 2-D PAGE and silver nitrate staining prior to the analysis of the differential protein expressions using image analysis software. Results Image analysis revealed obvious differential protein expressions of the cells in response to TPA treatment. Conclusion High-quality instruments for 2-D PAGE, skilled electrophoretic operation and efficient image processing are all essential in the reliable identification of stable functional proteins.