Objective Applying a reliable precise method to assess the mitotic index of cardiomyocytes, to disclose of disclosure the mechanism implicated in cardiomyocytes proliferation. Methods H9c2(2-1) cardiomyocytes were originally developed from rat BD1X heart (ATCC). These cells were cultured on coverslips. Double immunofluorescence staining with monoclonal antibodies (1:100) against phosphor-histone H3 and α-sarcomeric actin was performed on the cultured cells. Anti-mouse IgG FITC was used as the secondary antibody for the H3P antibody, and antimouse IgM Cy3 was used as the secondary antibody for the α-sarcomeric actin. DNA was visualized with Hochest 33342. All photographs were taken with an Olympus fluorescence microscope. Results The cytoplasm of cardiomyocytes appeared red, the mitotic chromosomes green with distinct shape, and Hochest 33342-stained nuclei blue.Conclusion Our method is the reliable and exact means to observe and assess cardiomyocy