Aim: The aim of the study was to investigate the effects of in vitro hemolysis on serum creatine kinase and creatine kinase MB isoenzyme activities with creatine kinase MB mass, troponin I and myoglobin measurements. Materials and methods: We prepared serum pools having analyte concentrations at normal and pathological values. Hemolysate was added into serum pools to obtain aliquotes with final hemoglobin concentrations of 21, 10.5, 5.25, 2.625, 1.312, 0.656, 0.328, 0.164, 0.08 and 0.041 g/L. Creatine kinase (by N-acetylcysteine-activated IFCC method) and creatine kinase MB activities (by immunoinhibition method) were measured in these pools. Creatine kinase MB (mass), troponin I and myoglobin concentrations were measured by chemiluminescence method. Mean percent changes were calculated and graphed as interferographs. Results: It was found that the positive interference due to hemolysis began to exceed the limit of 10% at lower Hb concentration for CK-MB activity than CK activity. When reference change value were considered, the critical effect of hemolysis began at higher hemoglobin concentrations. Hemolysis did not affect creatine kinase MB mass, troponin and myoglobin measurements when the limit of 10% change was considered. Conclusions: CK-MB activity is affected more profoundly than CK activity by hemolysis. Whereas CK-MB (mass), troponin I and myoglobin are absolutely not affected by hemolysis. © TurkJBiochem.com.