Drug-induced mitochondrial dysfunction can lead to severe adverse effects. Accordingly, new in vitro assay systems for assessing mitochondrial-related toxicity are required. Current systems evaluate drug-induced mitochondrial dysfunction based on cell death. However, if mitochondria are damaged without cell death, these methods run the risk of overlooking toxic or dangerous compounds. To solve this problem, we attempted to measure morphological changes semi-quantitatively by transmission electron microscopy and to detect subtle changes in mitochondrial function. To this end, we exposed HepG2 cells cultured in galactose-containing medium to benzbromarone (BBR), which impairs mitochondrial function. After 24 hr of BBR exposure, we compared the rate of cell death between galactose and glucose cultures. Before the onset of cell death, BBR increased the ratio of damaged mitochondria to a greater extent in galactose-cultured than glucose-cultured HepG2 cells. Our results suggested that this new in vitro assay system could detect mitochondrial-related toxicity before the onset of cell death.