Abstract: Objective：To analyze the specific peptide of cystatin C (CysC) and its characteristics by bioinformatics technology, and verify the predicted results by mass spectrometry. Methods：Online software was applied to analyze the physicochemical properties and homology of CysC peptides hydrolyzed by trypsin and predict the associated parameters of ionized fragmentation of specific peptide by mass spectrometry. Precursor ion scan and product ion scan were conducted on the samples of synthetic specific peptide. The recombinant human CysC and serum samples were analyzed by mass spectrometry after trypsin digestion. The results of analysis were compared with the outcomes predicted by bioinformatics. Results：T3 (ALDFAVGEYNK) was considered as the specific peptide of CysC by software analysis. When selecting［M+2H］²⁺ for product ion scan, almost all the y and b ions of fragmentation were observed using tandem mass spectrometry (MS/MS), showing consistency with Skyline predictions. Moreover, both the peptides from the human recombinant CysC and serum sample following the trypsin digestion were eluted at the same time with the isotopelabeled T3* under the fixed conditions. Conclusion：Bioinformatics technology could be available for picking out the specific peptides of target protein quickly and efficiently and predicting the ionized fragmentation precisely by mass spectrometry scanning.