The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine the K[subscript d] by plotting an absorbance ratio versus the concentration of free RP. The data are analyzed via a nonlinear fitting analysis to yield a K[subscript d] of 2.2 [plus or minus] 0.3 x 10[superscript -6] mol L[superscript -1]. This experiment illustrates how the K[subscript d]value can be determined and used to evaluate the strength of protein-ligand interactions. This experiment is appropriate for undergraduate students or first-year graduate students in biochemistry, chemistry, or physical chemistry. (Contains 2 figures.)