Abstract A size exclusion chromatographic method for studying ligand-macromolecule binding parameters is described. This equilibrium saturation method allows the determination of the concentrations of constituents in equilibrium and is specially useful for characterizing ligand-protein binding under conditions that can be compared with physiological conditions. The method has been used for measuring warfarin-human serum albumin (HSA) binding and for studying the influence of free fatty acids (FFA) and sodium dodecyl sulphate on warfarin-HSA binding. Some comparisons with the Hummel and Dreyer method are given. The influence of the FFA is strongly dependent on their chain length, with an inversion of the effect for a 10-carbon chain.