Pelitinib

High‐content screening (HCS) has become a powerful tool in drug discovery; however, its reliance on indirect readouts and surrogate markers limits HCS's ability to directly assess drug‐protein interactions at endogenous levels, particularly in subcellular contexts. Here, we report an approach to address these limitations by combining confocal imaging‐based HCS and bio‐orthogonal labeling with clickable probes. As a proof‐of‐concept, we synthesized a probe Triptolide‐alkyne (TL‐alk) that rapidly and specifically labels xeroderma pigmentosum type B (XPB), a critical protein in nucleotide excision repair (NER). Probe‐labeled XPB was conjugated to TAMRA to visualize the occupation of active sites, and EGFP and DAPI signals indicated XPB expression in the nucleus. Such a colorimetric HCS assay enabled the direct and precise measurement of drug occupancy rates in nuclear XPB of live cells. With this platform, pelitinib was identified as a novel ligand to bind XPB out of 1874 compounds containing. Food and Drug Administration (FDA)‐approved drugs. Pelitinib formed a covalent bond with cysteine residue 342 of XPB, suppressed XPB's ATPase activity, impaired NER, and synergistically enhanced chemotherapy. This study not only overcomes limitations of HCS, but also demonstrates the transformative potential of bio‐orthogonal labeling, such as in integration with HCS technologies, offering a novel framework for drug discovery targeting challenging protein systems.