The identification of a lead ligand from a library of compounds for a specific target requires both a selection process and a method to assess relative affinities. Using a tandem-column chromatographic technique, we have developed a novel and rapid method for determination of relative affinities for ligands binding to a specific target molecule. We demonstrate, using known ligands for the lipid A region of lipopolysaccharide, that the relative affinities of these ligands can be determined and may be used to characterize the competitive interaction between ligands for the same target. The method can be adapted toward screening of soluble libraries of peptides and small molecules and those ligands exhibiting a desired affinity can be rapidly selected for further characterization/development.