We developed a two-step ELISA to determine the immunoreactive fraction of monoclonal antibodies in conditions of antigen excess. An antibody aliquot at limiting dilution was incubated in wells coated with increasing amounts of antigen up to concentrations that bind 100% of antibody. At equilibrium, a supernatant aliquot was transferred to a second plate coated with excess of antiglobulin, and the captured antibody was incubated with peroxidase-conjugated anti-IgG. Antibody was quantitated from the enzyme velocity gradient in a kinetic ELISA, and the immunoreactive fraction calculated as (1 - gradienti/gradientT) x 100, where i and T are the gradients for the free and total antibody fractions. For four distinct monoclonal antibodies (anti-diphtheria toxoid, −cholera toxin, −bovine serum albumin (BSA), and -trinitrophenyl-BSA), measurement of inter-assay variability yielded values ranging from 3.1 to 7.4 (% coefficient of variation), which supports method repeatability. This ELISA is simple, precise, and applicable to mono- and polyclonal antibodies. •Antibody quantitation by measuring initial velocity of enzyme product formation.•Initial rate measurements ensure ELISA velocity proportional to analyte concentration.•A mAb isotype-matched standard curve could reduce antiglobulin heterogenous reactivity.