Multiplexed microsphere-based immunoassays can be developed to simultaneously measure multiple analytes in a biologic system by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. A multiplexed sandwich immunoassay is based on an ELISA format that is transferred directly to microspheres to quantitate multiple antigens. These assays require smaller sample volumes, are less expensive, and are as reproducible, reliable, and sensitive as ELISAs. However, potential cross-reactivities between multiplexed antibodies, antigens, and specimens need to be systematically eliminated during the validation process. Sandwich and competitive immunoassays, which require only one antigen-specific antibody, can be combined in the same multiplexed array. Antibody-capture immunoassays are used to detect multiple antibodies from a specimen for diagnostic or surveillance purposes. The protocols for these three multiplexed immunologic assays are accompanied by methods for coupling analytes to microspheres and biotinylation of antibodies with a water-soluble derivative.