The protein components contained in the snake venom of Gloydius shedaoensis shedaoensis（GSS-SV） were labeled with the fluorescent dye of Cy5.Then,the two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis（2D SDS-PAGE） profile of GSS-SV proteome was obtained.Gel image analysis by DeCyder software resolved over 1 000 protein spots with the apparent molecular weights ranging between 10 and 150 ku on the gel.The protein spots with the pI ranging between 4 and 7 covered about 78.8% of the overall protein spots detected by the 2D SDS-PAGE.Post-staining of the gel was performed by a high sensitive fluorescent dye,Deep Purple.Five protein spots were excised from the gel and digested by trypsin in-gel.The tryptic peptides were separated by high performance liquid chromatography（HPLC） and subsequently sequenced by electro-spray ionization tandem mass spectrometry（ESI-MS/MS）.The mass spectrometry data were searched against the NCBInr database through the software of Sequest Bioworks for protein identifications.The above mentioned five gel spots were identified as the snake venom L-amino acid oxidase,metalloproteinase,thrombin-like enzyme（salmobin）,plasminogen activator and phospholipase A₂,respectively.The results prove that the proteomic techniques based on two-dimensional electrophoresis,protein fluorescent labeling technique and HPLC-ESI-MS/MS can be utilized in the analysis of protein components in the snake venom complex at high throughput level.