AbstractOne of the most important indicators of myocardial regeneration is cardiomyocyte proliferation. However, it is difficult to distinguish cardiomyocytes in the regenerating stage from binucleated or multinucleated myocytes by conventional morphometric techniques. As cell cycle progression (CCP) scores have been successfully applied to the evaluation of the proliferation of cancer cells, we sought to establish a multi‐gene score to evaluate cardiomyocyte proliferation in this study. Given the disturbances of nuclear division without cell division that occurs in cardiomyocytes, ten cytokinesis‐correlated genes (Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2, and CDC42) were chosen to establish the cardiomyocyte cytokinesis score (CC‐Score). The expression levels of these genes in H9C2 rat cardiomyoblast cells, the proliferation of which were stimulated or inhibited, were detected using qRT‐PCR. To confirm the feasibility of the CC‐Score system, four conventional methods for evaluating cardiomyocyte proliferation, including the MTT assay, BrdU assay, immunofluorescence, and flow cytometry analysis, were used in each group. The results of the CC‐Score in the assessment of the proliferation of H9C2 cells were consistent with those of four commonly used proliferative assay methods. We conclude that the CC‐Score can be used to assess the proliferation status of H9C2 cells, and suggest that the CC‐Score may be a potential method for the assessment of cardiomyocyte proliferation in myocardial regeneration. However, validation studies utilizing primary cultured rat cardiomyocytes and heart tissue are warranted.