A simple method was developed for the rapid characterization of the covalent binding of haptens to proteins such as enzymes, bovine serum albumin (BSA), and other carrier-proteins and antibodies. In the present study, a commercially available fentanyl-BSA conjugate was characterized by a 4'-hydroxyazobenzene-2-carboxylic acid (HABA) dye assay that followed a biotinylation reaction. This protocol allowed the indirect observation of the average hapten number per BSA molecule. Such measurement is useful for optimizing reaction conditions to yield a more precisely defined product for immunological applications. The obtained result was within the limits suggested by the manufacturer of the conjugate.