Doxorubicin (Dox) belonging to an anthracycline class-anti cancer antibiotic exhibits superior clinical outcome for the anti-cancer regimen; however, its use also causes severe cardiomyopathy by impairing mitochondrial function. Mitochondria maintain their function utilizing fission-fusion cycle and dysfunctional mitochondria should be removed by mitophagy. We have reported that Dox affected the mitochondrial fission-fusion cycle to a fission-dominant and caused the mitochondrial dysfunction; however, the effect of Dox on the mitophagy is unclear. In this study, we applied a unique fluorescent protein, Keima Red, with a mitochondrial targeting sequence to evaluate the effect of Dox on mitophagy in rat cardiomyoblast, H9c2 cells. Keima Red emits the fluorescence at 620 nm with the excitation wavelength at 440 nm in the neural pH such as cytosol, whereas, the excitation wavelength is shifted to a longer wavelength at 550 nm in the acidic condition like in the lysosome. When the cells were treated with a mitochondrial uncoupler agent, CCCP, the excitation wavelength was shifted from 440 nm to 550 nm indicating that mitophagy was induced. In contrast, the excitation wavelength of Keima Red was dominant at 440 nm than that of 550 nm following administration of 1 µM Dox suggesting that Dox did not elevate levels of mitophagy and the dysfunctional mitochondria retained in the cytosol but not in the lysosome. We further measured the levels of reactive oxygen species (ROS) production and the cell viability. Dox-administration significantly augmented the ROS-production and thus, resulted in the cause of cell death. These results suggest that Dox inhibits the mitophagy that is essential for the clearance of dysfunctional mitochondria and the Dox-caused impairment of mitophagy may be one of the causes of cardiomyopathy.