A very simple method to determine the activity of AST isoenzymes by using the AST inhibitor AMANO-3 has been developed. This inhibitor is a protease which selectively proteolyzes m-AST in the sample and no influence to the activity of c-AST. The coefficient of variation. of the proteolytic method is among 3.5%～7.9%. Resultsobtained by the present method correlated with those by the electrophoresis method. In the present assay system, reference values for mitochondrial AST activity in healthy people ranges from 1.64～9.64 U/L and the reference values for cytosolic AST activity in healthy people range from 5.55～16.85 U/L. The study showed that the selective measurement of AST isoenayme method is a rapid and accurate method. It has great clinical significance.