A very simple method to determine the activity of LD isoenzyme Ⅰ by using αchymotrypsin and guanidine has been developed.LD-3～LD-5 can be completely inactivated and LD-2 can be partially inactivated byαchymotrypsin.Guanidine can further completely inactivate LD-2. This method has been used in automatic analyzer. The coefficient of variation of the proteolytic method is among 2.7% ～4.4%.Results obtained by the present method correlates with those by the electrophoresis method (r=0.992, y=0.967x-1.957). In the present assay system, the reference value for LD-1 activity in healthy people ranges from 29.78～59.26 U/L. The study showed that the selective measurement of LD-1 isoenzyme method is rapid and accurate. It has great clinical significance.