In accordance with requirements of the ICH S7B safety pharmacology guidelines, numerous next-generation cardiotoxicity studies using human stem cell-derived cardiomyocytes (CMs) are being conducted worldwide. Although several stem cell-derived CMs are being developed for commercialization, there is insufficient research to verify whether these CMs can replace animal experiments. In the present study, in vitro high-efficiency CMs derived from human embryonic stem cells (hESC-CMs) were compared with Sprague-Dawley rats as in vivo experimental animals, and primary cultured in vitro rat-CMs for cardiotoxicity tests. In vivo rats were administrated with 2 consecutive injections of 100 mg/kg isoproterenol, 15 mg/kg doxorubicin, or 100 mg/kg nifedipine, while in vitro rat-CMs and hESC-CMs were treated with 5 μM isoproterenol, 5 μM doxorubicin, and 50 μM nifedipine. We have verified the equivalence of hESC-CMs assessments over various molecular biological markers, morphological analysis. Moreover, we have identified the advantages of hESC-CMs, which can distinguish between species variability, over electrophysiological analysis of ion channels against cardiac damage. Our findings demonstrated the possibility and advantage of high-efficiency hESC-CMs as next-generation cardiotoxicity assessment.