Drug repositioning saves the time and cost of drug development. It is a promising approach reducing risk of side effects because it starts from already approved drugs. In this study, reverse chemical proteomics enables access to new targets and pathways of existing drugs targeting human diseases. Artesunate (ART) is used as a case compound in this study. ART is the most potent and safe antimalarial drug which is one of the derivatives of the Artemisinin, the Nobel Prize winning compound in 2015. The unbiased interrogation of several human cDNA libraries, displayed on bacteriophage T7, identified a high affinity human target of ART; the Bcl-2 antagonist of cell death promoter (BAD). ART inhibits the phosphorylation of BAD, promoting the formation of the BAD/Bcl-xL complex and the subsequent intrinsic apoptosis. Direct interaction of ART with BAD was validated by DARTS and ICC experiments using label-free ART and fluorescence-ART, respectively. In addition, ART induces caspase-dependent apoptosis by triggering release of cytochrome c from mitochondria to cytosol in cancer cells. Collectively, this case study of reverse chemical proteomics demonstrates the potential of this method for new target identification of known clinical drug in drug repositioning.