Doxorubicin (Dox) has been used as an effective antitumor agent against various types of cancers. However, its application is limited owing to severe cardiomyopathy. Many researchers have attempted to clarify the mechanism of DOX-induced cardiomyopathy; however, its underlying mechanisms remain unclear. We showed that Dox impaired autophagic flux. In this study, we focused on the effect of Dox on lysosomes, a prerequisite organelle for autophagy that degrades autophagic cargoes. We speculated that Dox might inhibit autophagy by inhibiting the lysosomal function. To evaluate the effect of Dox on lysosomal acidification, we examined the effect of Dox on lysosomal acidification by staining cells with the acidophilic fluorescent dye acridine orange to determine the pH of lysosomes. Acridine orange emits red fluorescence under acidic conditions, whereas it emits green fluorescence under neutral pH conditions. The green and red fluorescence intensities of acridine orange were measured by flow cytometry. Bafilomycin, an H+-pump inhibitor of lysosomes, significantly inhibited lysosomal acidification. In contrast, lysosomal acidification increased considerably after Dox treatment. We further explored the effect of Dox on the expression of transcription factor EB (TFEB), which is known to regulate lysosomal function. Unexpectedly, Dox did not affect the expression of TFEB. These results indicated that Dox does not affect lysosomal acidification or function.