Abstract: Objective To explore an optimal cartilage protein extraction approach that can guarantee both the image quality and the result fidelity of the two-dimensional gel electrophoresis (2-DE) technique. Methods Knee cartilage samples were obtained from femoral condyles (n=17). Approaches used for protein samples of 2-DE were grouped: (1) Extracting protein directly from cartilage samples (Cartialge approach); (2) Total protein was treat with cetylpyridinium chloride (CPC) after being extracted from cartilage samples (Cartilage plus CPC approach); (3) Extracting protein from chondrocytes directly isolated from cartilage samples (directly extracted chondrocytes approch). (4) Extracting protein from cultured chondrocytes (cultured chondrocyters approach). Image qualities generated by 2-DE with different protein extracting approaches were compared and the capabilities of these approaches in generating believable proteomic results were also evaluated. Results Isoelectrofocusing was failed in the cartilage approach group. Cartilage plus CPC precipitation approach group can reach isoelectrofocusing, but the protein spots it generated were much less than those of both the chondrocyte approaches. The directly isolated chondrocytes approach group generated comparable high-quality gel images to that of the cultured chondrcoytes approach group. It also isolated some protein spots that were missed in the high-molecular or alkaline regions that of the cultured chondrocytes approach group. Results of mass spectrography revealed that the proteins located in these regions were collagen VI, TGF-β2 and annexin, etc. All were pathologic proteins correlated with the pathogenesis of osteoarthritis. Conclusion The approach of isolating chondrocytes from cartilage and extracting total protein directly for 2-DE analysis can avoid the problem of isoelectrofocusing disturbance and generate believable results through avoiding altered protein expression spectra under cell-culture environment, which was an optimal protein extraction strategy for 2-DE technique in investigations on cartilage-related diseases.