UV-vis spectrophotometry has been applied to determine acid dissociation constants of the prenylated chalcone xanthohumol. The pKa values were compared with those derived from pH-metric titrations. The order of the deprotonation site in the xanthohumol molecule was estimated by quantum mechanical calculations as 2'-OH, 4'-OH, and 4-OH. Furthermore, the electronic and spectroscopic properties of xanthohumol have been investigated on the basis of the time-dependent density functional theory (TDDFT). The TDDFT method, combined with a hybrid exchange-correlation functional using the B3LYP and CAM-B3LYP levels of theory in conjunction with the SMD solvation model, was used to optimize all geometries and predict the excitation energies of the neutral form and ionized species of the chalcone depending on pH value. The computed results were in good agreement with the experimental data. Consideration of the acid-base profile in conjunction with other molecular properties has a great importance and has the potential to be used to further improve the bioavailability of xanthohumol.