Spectroscopic analyses reveal that acridone (AD) penetrates through the structure and enters the hydrophobic cavity of the protein beta-lactoglobulin (beta LG). Although the protein contains two tryptophan (Trp) residues, AD interacts with only one (Trp-19), which is authenticated by the appearance of a single isoemissive point in TRANES. Alteration in the secondary structure of the protein while AD pierces through beta LG is evident from the circular dichroism spectroscopic study. The ground-state interaction between AD and beta LG is proven from the UV-vis spectroscopic study and the static nature of quenching of intrinsic fluorescence of the protein by the ligand. The steady-state fluorescence study in varied temperatures indicates the involvement of hydrogen bonding in the ligand-protein interaction. Further, the time-resolved fluorescence anisotropy study gives a hint of the presence of a hydrogen bond in AD-beta LG interaction, which possibly involves the rotamers of Trp-19. In fact, the idea of involvement of rotamers of Trp-19 is obtained from the increase in fluorescence lifetime of beta LG in the presence of AD. The docking study agrees to the involvement of hydrogen bonding in AD-beta LG interaction. The direct evidence of hydrogen bonding between Trp and AD is obtained from the laser flash photolysis studies where the signature of formation of ADH(center dot) and Trp(center dot) through hydrogen abstraction between Trp and AD, loosely bound through hydrogen bonding, gets prominence. Thus, binding of AD to beta LG involves hydrogen bonding in a hydrophobic pocket of the protein.