Cardiac dysfunction is a major side effect caused by anticancer drug treatment. Doxorubicin, an anthracycline-derivative anticancer drug, exhibits dose-dependent cardiotoxicity, which limits its clinical use. Cardiotoxicity ranges from decreased ejection fraction, arrhythmias, to highly symptomatic congestive heart failure, which are all associated with high mortality. Cardiomyocytes require energy to beat and therefore retain an abundance of mitochondria. We established quantitative measurements of mitochondrial length and respiratory activities, and contractile functions using cardiomyocytes. We found that exposure of human iPS cell-derived cardiomyocytes to anticancer agents suppressed myocardial contraction and enhanced mitochondrial hyperfission. The oxygen consumption rate was significantly reduced. Knockdown of dynamin-related protein 1 (Drp1), mitochondrial fission-accelerating GTP-binding protein, suppressed mitochondrial hyperfission and cytotoxicity caused by anticancer agents. This indicates that visualizing mitochondrial functions in human iPS cell-derived cardiomyocytes will be helpful to assess the risk of cardiotoxicity caused by anticancer agents, and that the maintenance of mitochondrial quality will become a new strategy to reduce anti-cancer drug-induced cardiotoxicity.