Cisplatin (CDDP) is a first-line chemotherapeutic drug for treating various cancers. However, CDDP also damages normal cells and causes many side effects. Recently, CDDP has been demonstrated to kill cancer cells by targeting mitochondria. Protecting mitochondria might be a potential therapeutic strategy for CDDP-induced side effects. β-Lapachone (β-lap), a recognized NADIn this study, the protective effects of β-lap on mitochondria against CDDP cytotoxicity in normal cells were evaluated.In vitro cell models were used in this study, including 3T3 fibroblasts, human dermal fibroblasts, MCF-7 breast cancer cells, and MDA-MB-231 breast cancer cells.Cells were treated with CDDP and β-lap, and cell survival, NADThe results demonstrated that β-lap significantly decreased CDDP cytotoxicity in normal fibroblasts. With various inhibitors and siRNAs, β-lap reduced CDDP-induced damage to normal fibroblasts by maintaining mitochondrial activity and increasing autophagy through the NQO1/NADβ-lap could be a good synergistic drug for reducing the side effects of CDDP without affecting the anticancer drug effect.