Hesperidin is a flavanone glycoside widely available for dietary intake in citrus fruits or citrus fruit derived products; however, exhaustive and reliable data are scarcely available for biological activity when it exerts protective health effects in humans. The principal intent of this work is to check binding domain and structural changes of human serum albumin (HSA), the primary carrier of flavonoids, in blood plasma association with hesperidin by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) methods. From molecular modeling simulations, subdomains IIA and IIIA, which correspond to Sudlow's sites I and II, respectively, were earmarked to possess affinity for hesperidin, but the affinity of site I with flavanone is greater than that of site II. This corroborates the site-specific probe and hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results placing the hesperidin at warfarin-azapropazone and indole-benzodiazepine sites. Steady state and time-resolved fluorescence manifested that static type, due to HSA-hesperidin complex formation (1.941 × 10(4) M(-1)), is the operative mechanism for the diminution in the tryptophan (Trp)-214 fluorescence. Moreover, via alterations in three-dimensional fluorescence and CD spectral properties, we can securely draw the conclusion that the polypeptide chain of HSA is partially destabilized after conjugation with hesperidin. We anticipate that this study can provide better knowledge of bioavailability such as absorption, biodistribution, and elimination, of hesperidin in vivo, to facilitate the comprehension of the biological responses to physiologically relevant flavanones.