An enzyme-linked immunosorbent assay to measure affinity constants of hapten-specific monoclonal antibodies has been developed. In this method an antibody-immobilizing format is used. Antibody affinity toward hapten-enzyme tracer is first calculated by using Scatchard’s method. Using this affinity, antibody-hapten affinity is then calculated by following the principles of chemical thermodynamics and mass conservation. The affinity from this method agrees with the IC50 values in ELISA. This method is useful in early determination of affinity during monoclonal antibody development. Compared with the antigen-coated format, this method avoids the problem of distinguishing binding of unliganded antibody and singly liganded antibody to antigens on solid phase.