The binding of diflunisal to hydroxypropyl-β-cyclodextrin (HPβCD), bovine serum albumin (BSA), human serum albumin (HSA), normal human plasma, and mixed solutions of HPβCD/ protein was studied at 25°C, pH 7.4, by potentiometry using an electrode selective to diflunisal. The experimental data for diflunisal/ HPβCD fit well to the 1:1 binding model. The binding of diflunisal with each of the studied proteins was compatible with a model having two independent classes of binding sites. The binding of diflunisal in mixed solutions HPβCD/BSA, HPβCD/HSA, and HPβCD/plasma increased considerably when the HPβCD concentration was increased. The binding behavior of the two biomolecules in the mixed solutions of HPβCD/BSA or HPβCD/ HSA was described with an “additive” model formulated on the basis of the estimates of the binding parameters of diflunisal derived from the separate experiments with each one of the binders tested. The lower than theoretical binding observed in HPβCD/plasma solutions was ascribed to the competitive displacement of diflunisal from the HPβCD cavity by plasma cholesterol.