Altered glucose-6-phosphate dehydrogenase (G6PD) status is influential in many cellular pathophysiological processes and diseases, making G6PD a potential target for cancer therapy. However, the available G6PD inhibitors are very limited and restricted. Here we developed a reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) absorption photometry assay based on enzyme kinetics to characterize G6PD activity. In this way, we performed a high-throughput screening (HTS) to an in house library. And then we identified compound named Wedelolactone inhibiting G6PD strongly in a non-competitive, reversible way. In addition, we did the surface Plasmon Resonance (SPR) assay and indicated the K