We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.