The modulation of ligand p"K"[subscript a] due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the p"K"[subscript a] of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an experiment using spectrophotometric method to demonstrate how ligand p"K"[subscript a] values can be influenced by specific interactions in the protein-binding pocket using riboflavin binding protein (RP) and its ligands (riboflavin, RF, and neutral red, NR). A direct plot of observed absorbance versus pH was analyzed by nonlinear regression. The p"K"[subscript a] values of free and RP-bound RF were determined to be 10.0 plus or minus 0.1 and approximately 13.3, respectively, and the p"K"[subscript a] values of free and RP-bound NR were 6.8 plus or minus 0.1 and 7.8 plus or minus 0.1, respectively. This laboratory clearly demonstrates that the environment of a protein-binding site can affect the p"K"[subscript a] value of a ligand. The experiment can be adapted or used as-is for undergraduate students in biochemistry or chemistry (analytical or physical chemistry) or first-year graduate students in biochemistry and related fields. (Contains 2 figures and 1 scheme.)