Abstract: In order to establish an effective analytical method to detect the key proteases which affect the metabolism and the plasma half-life of peptides in vivo, a method to analyze the key proteases of peptide drugs based on high performance liquid chromatography in vitro was established. The general enzymatic reaction conditions in vitro were as follows: pH was 7. 8 or 9. 0 and the buffer system was 0. 01 mol/L PBS or 50 mmol/L Tris buffer. The results of pramlintide detected by this method showed that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 had the strongest hydrolysis on pramlintide. The result was consistent with that determined by microscale thermophoresis, which indicated that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 were the key proteases of pramlintide. This analytical method provides the basis for high-throughput stability screening of peptides and can be used to analyze key proteases of peptide drugs. It can also provide guidance for optimizing the protease stability of peptide drugs.