A multiplex assay for the quantitation of immunoglobulin G (IgG) serum antibodies directed against Clostridium tetani toxin (TT), Corynebacterium diphtheriae toxoid (DTxd), and the Bordetella pertussis antigens pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn) was developed on an Evalution® platform to enhance the evaluation of the specific antibody response towards protein antigens in suspected humoral immunodeficiencies. Evalution® is a microfluidic and microparticle-based platform with the possibility to analyse single samples and to perform real-time kinetic measurements of antibody binding. All individual antigens were covalently linked to the carboxylated microparticles after which samples and fluorescently labelled detection antibodies were flowed over the microparticles in the microfluidic channels of the assay cartridges of the system. The developed assay showed very good sensitivity, specificity, and intra- and inter-assay coefficients of variation (CVs for the different antigens between 1.72-3.53% and 3.54-5.79%, respectively). Furthermore, the correlation of the Evalution pentaplex with a Luminex pentaplex using a panel of 48 human serum samples was excellent, with Spearman correlation coefficients between 0.936 for PT and 0.982 for DTxd (p < 0.0001 for all). Finally, we showed in a proof-of-concept experiment the potential of the Evalution® platform to simultaneously measure concentrations and binding kinetics (as a surrogate for avidity) of the IgG antibodies to the selected protein antigens. Overall, these findings show that this new Evalution pentaplex can accurately measure the antibody response to TT, DTxd, PT, FHA and Prn. It also has the potential to measure antibody binding and dissociation kinetics.