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The reactivity-based screening (RBS) approach was developed.Thiol probe targeted withanolides via the nucleophilic addition to 2(3)-en-1-one.Eleven cytotoxic withanolides were discovered from plant extract directed by RBS.The reactivity-based screening (RBS) was developed for directed discovery of cytotoxic withanolides. In this study, a thiol probe, 4-chlorobenzenethiol, was used to selectively attack cytotoxic withanolides containing potential pharmacophore, 2(3)-en-1-one in ring A (AEO) and 5β,6β-epoxy in ring B (BE), from the plant extract of Physalis angulata var. villosa. The screening was performed based on the potential mechanism of 4-chlorobenzenethiol nucleophilic addition to AEO, followed by detection of adducts using liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS). Guided by RBS, eleven target withanolides, including five new compounds, physagulides R-V (10–14) and six known ones (2, 7–9, 15, 16) were discovered. All of them exhibited cytotoxicity against the both tested cell lines, especially, compounds 2, 7, 8 and 14 showed potent activities with IC50 values of 1.57–6.29 μM. The results suggested that RBS was efficient and accurate for rapid identification of cytotoxic withanolides and could guide isolation of target components from the complex medicinal plant extract.