Abstract: The proteases secreted by strain BS5582 were rapidly identified by zymogram method in this study.The highest extracellular protease activity was detected under neutral condition. The three peaks of protease during fermentation process indicated that BS5582 might secrete several proteases.The zymography displayed five clear bands of casein hydrolysis on the gel. Protein bands on the non- denaturing- SDS- PAGE gel corresponding to those on the zymography gel were identified by mass spectroscopy. The results showed that strain BS5582 secreted three kinds of proteases.They were Bacillopeptidase F ( Bpr) , extracellular neutral metalloprotease ( Mpr) and Subtilisin Bpn '. Bpr and Mpr had two forms in the medium, respectively. The different forms of extracellular proteases secreted by BS5582 could be rapidly identified by the zymography method combined with application of mass spectrometry.Therefore, it was an efficient and rapid method to identify mixture of proteases.