Protein C is an anticoagulant protein that circulates in blood as a zymogen of a serine protease. Recently the exogenous Protein C activator has been obtained from the venom of Agkistrodon Contortrix Contortrix. We also reported a functional assay method which activates Protein C with PROTAC and then measure the amidolytic activity with chromogenic substrate (S-2366) by COBAS FARA. At present, S-2366 (Glu-Pro-Arg-pNA), CBS 65-25 (Lys-Pro-Arg-pNA) and SPECTROZYME PCa (Lys-Pro-Arg-pNA) are used as the chromogenic substrate for activated Protein C. We studied which substrate is more suitable for amidolytic activity assay for Protein C by PROTAC in 99 patients under long-term Warfarin therapy and 29 as normal subjects. Our results indicate that SPECTROZYME PCa and CBS 65-25 are unsuitable for Protein C activity assay with PROTAC. Because change of absorbance was detected in Protein C deficient plasma and we couldn't gain good correlation between amidolytic activity of activated Protein C measured with these chromogenic substrates and antigenicity of Protein C by EIA. On the other hand, S-2366 is the most specific for activated Protein C and we also obtained good correlation against Protein C antigen.