Abstract: 【Abstract】 Objective To investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes. Methods We employed three different digestion methods to separate neonatal rat cardiomyocytes. Group A was 0.08% trypsin digestion alone，group B was 0.08% trypsin+0.08% type Ⅱ collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type Ⅱ collagenase isolated digestion. The number of cells and cell viability after differential adhesion were recorded. The purity of cardiomyocytes was evaluated by immunofluorescence staining. The cell vitality was assessed by the detection of mitochondrial membrane potential with JC-1 staining, and the ratio of red to green fluorescence intensity by laser scanning confocal microscope. Results _{There was no}statistically significant difference in the number of cells between three groups （P >0.05）.The rate of cell viability in group C was significantly higher than that in group A （P <0.01）. No statistically significant difference was found in the purity of cardiomyocytes between three groups （P >0.05）. The ratio of red to green fluorescence intensity in group A, B and C were 0.928±0.078, 0.943±0.099 and 1.160±0.089, respectively; the ratio in group C was significantly higher than that in group A and group B (P <0.01). Conclusion The cell isolation method with 0.08% trypsin and 0.08% type Ⅱ collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.