Napabucasin (NAPA) has been shown as a potent cancer stemness inhibitor that has demonstrated promising activity towards cancer of different types in early phase clinical trials despite satisfactory results in phase III trials. Unfortunately, two recent phase III NAPA clinical trials failed to meet the primary endpoint of overall survival. The reason for the failure is not clear, but possible ways to rescue the clinical trial are to stratify patients with biomarkers that could predict NAPA response, and/or to seek alternative strategies to increase the NAPA efficacy. Here, we report the identification of NAD(P)H dehydrogenase 1 (NQO1) as a major determinant for NAPA efficacy by a proteomic approach. In vitro evidence showed that NAPA is a substrate for NQO1, which mediates the generation of ROS for cell death. We also demonstrate that inducing NQO1 expression by pre-exposure to IR or activation of its transcription factor NRF2 by drugs, including one approved FDA, can increase the cytotoxicity of NAPA. Our findings suggest the use of a companion diagnostic test to identify tumors with high NQO1 expression in future NAPA trials and the strategies for expanding the application of NAPA-based regimens for human cancer therapy.