Objective To investigate the interaction mechanism and structure-activity relationship between two hop-derived small molecules different in structure (α-acid and xanthohumol) and barley protein Z.Methods α-acid and protein Z with high purity are prepared through solvent extraction and anion exchange column, respectively. The interaction between α-acid/xanthohumol and protein Z, as well as the conformational change of protein, is characterized by multi-spectral analysis. Finally, the interaction mode and molecular binding mechanism between α-acid/xanthohumol and protein Z are further investigated by molecular dynamics simulation.Results Both α-acid and xanthohumol can effectively quench the intrinsic fluorescence of protein Z, with α-acid inducing a red shift and xanthohumol causing a blue shift in fluorescence spectra. Additionally, the binding stoichiometry for both molecules approaches 1∶1. The molecular dynamics results reveal that hydrogen bonds and hydrophobic interactions are the main forces between the two hop-derived small molecules and protein Z.Conclusion Xanthohumol exhibits a stronger affinity toward protein Z compared to α-acid.