ScopeMany phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,β‐unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell‐protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently.Methods and resultsHere we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone‐containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN‐alkyne), we purified covalent protein‐electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose‐6‐phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations.ConclusionWe find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.