Abstract: Objective To assess the advantages and disadvantages of several synovial fluid preparation methods for two-dimensional gel electrophoresis in patients with knee osteoarthritis (KOA). Methods Eight synovial fluid samples from KOA patients admitted to our hospital from January 2012 to March 2012 for arthroscopic diagnosis and treatment were mixed up and divided into 36 equal shares. Of which, 9 were not pretreated, 9 were treated with hyaluronidase, 9 with affinity chromatography to deplete highabundance proteins, and 9 with combined hyaluronidase and affinity chromatography. The 36 equal sample shares were treated by two-dimensional gel electrophoresis. Different gel electrophoresis images of the treated and untreated samples were compared to determine the optimal pretreatment plan which was used in pretreatment of synovial fluid from 12 KOA patients. Differentially expressed proteins were detected and identified by mass spectrometry. Results The samples showed the best gel electrophoresis images, and the joint disease–related protein factors apolipoprotein and fibrinogen fragment D were identified in the synovial fluid from 12 KOA patients after the samples were treated with combined hyaluronidase and affinity chromatography. Conclusion Samples pretreated with combined hyaluronidase and affinity chromatography display high-quality gel electrophoresis images, and can thus be used as a first choice of pretreatment in proteomic study.