Drug-plasma protein binding may greatly influence the bioavailability and metabolism of a plasma-borne drug, the bound form being partially protected from the metabolic fate of the unbound drug. Traditionally, equilibrium values (e.g., percentage binding) for drug-protein binding have been measured to rationalize in vivo phenomena. However, such studies overlook the influence of kinetics. A rapid method of simultaneously determining kinetic rate constants and equilibrium constants from chromatographic profiles has been developed, based on the use of immobilized protein columns and HPLC. By measuring the chromatographic profiles (the position and width) of a retained and an unretained compound one can directly determine both the rate and equilibrium constants. Results are presented for the binding of L-tryptophan to human serum albumin to exemplify the method. The association equilibrium constant (Ka) and the association and dissociation rate constants (k(a) and k(d), respectively) were thereby measured in an aqueous pH 7.4 environment at 37 degrees C as 0.84 10(4) M(-1), 5.8 10(4) M(-1) s(-1), and 6.9 s(-1), respectively. These compare favorably with previously published results. The described method may be used in quantitative structure-property relationship-based rational drug discovery or for the rationalization of drug pharmacokinetics.