Abstract: OBJECTIVE To explore the application of HL60-IL6 method in the pyrogen detection for collagenase and urokinase for injection. METHODS According to the general rule 1193 of Chinese Pharmacopoeia(the 2015 Edition), the minimum effective dilution concentration of collagenase and urokinase for injection was determined. The max concentrations of samples which had no effect on HL60 proliferation, pyrogen test and IL-6 test were determined by the methods of proliferation-inhibition test, pyrogen interference and IL-6 interference test. According to the results, the in vitro pyrogen test for collagenase and urokinase for injection was established. Then, the pyrogen of 2 batches of collagenase for injection and 19 batches of urokinase for injection were determined with the method. RESULTS For collagenase, the minimum effective dilution concentration was 0.04 U·mL^-1, the sample which was ≤ 60 U·mL^-1 had no effect on HL60 proliferation, and ≤ 6 U·mL^-1 had no effect on pyrogen and IL-6 test. For urokinase, the minimum effective dilution concentration was 100 U·mL^-1, the sample which was ≤ 1 000 U·mL^-1 had no effect on HL60 proliferation, and ≤ 500 U·mL^-1 had no effect on pyrogen and IL-6 test. As a result, the suitable concentration was 6 U·mL^-1 for collagenase and 500 U·mL^-1 for urokinase respectively. Then, the suitable the in vitro pyrogen test for collagenase and urokinase for injection was established. There were no pyrogen in all samples. CONCLUSION The method is suitable for in vitro pyrogen test of collagenase and urokinase for injection.