Bead-based suspension arrays built with multiple antigens enable detection and identification of specific antibodies in human and animal serum. Here we describe how to use synthetic peptides as antigens in a serological Luminex assay. Biotinylated peptides immobilized on avidin beads are used to capture serum antibodies, which in turn are detected using a generic immunoglobulin-binding protein or broad anti-bird antibodies in resp. mammalian and avian sera. Background is suppressed by pre-blocking sera and by using a blocking agent during antibody capture. By employing an internal negative control, serum-specific cutoffs can be determined and/or signals can be normalized. The provided protocols allow creating multiplex bead-based suspension arrays using peptides as antigens to capture and detect specific serum antibodies from mammalians, including humans, and avian species.