We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic two color fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (similar to 29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (t). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (K-D) as 5.42 X 10(4) M-1 and the association constant (K-s) as 5.59 X 10(34) M-1. Time-resolved fluorescence anisotropy decay studies show dramatic (similar to 170 times) increase in the rotational correlation time (pi(ro)t), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.