Abstract Specific protein-binding reactions were monitored utilizing ligand-fluorescent dye conjugates which were substrates for porcine esterase. Biotin was coupled directly to umbelliferone through an ester bond, while 2,4-dinitrophenyl derivatives were conjugated to fluorescein via an ester linkage. Hydrolysis of these nonfluorescent esters with an esterase yielded fluorescent products, and the reaction rate was related quantitatively to the conjugate concentration. When the conjugated ligands were bound to their specific proteins (avidin or antibody to the dinitrophenyl residue), they were inactive as substrates. This assay allowed the measurement of unbound conjugate without separation from protein-bound conjugate. The inactivation of the conjugated substrates was relieved by the addition of free (unconjugated) ligand in competitive binding reactions. In addition to measuring free ligand levels, this assay was used to detect specific binding proteins, e.g., during the purification of antibody to the dinitrophenyl residue.