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Here, we report an approach to address these limitations by combining confocal imaging\u2010based HCS and bio\u2010orthogonal labeling with clickable probes. As a proof\u2010of\u2010concept, we synthesized a probe Triptolide\u2010alkyne (TL\u2010alk) that rapidly and specifically labels xeroderma pigmentosum type B (XPB), a critical protein in nucleotide excision repair (NER). Probe\u2010labeled 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)\u2010approved 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\u2010orthogonal labeling, such as in integration with HCS technologies, offering a novel framework for drug discovery targeting challenging protein systems.<\/jats:p>","DOI":"10.1002\/anie.202505585","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T09:34:00Z","timestamp":1751362440000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Imaging\u2010Based High\u2010Content Screening with Clickable Probes Identifies XPB Inhibitors"],"prefix":"10.1002","volume":"64","author":[{"given":"Shuqi","family":"Li","sequence":"first","affiliation":[{"name":"Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University  Hung Hom Hong Kong SAR P.R. 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