Peptide microarrays are useful instruments for miniaturized high-throughput, high-content immunoassays. The substitution of linear epitopes of the protein antigen with short, synthetic peptides is virtually a straightforward approach to capture antigen-specific antibodies from serum samples; however, both the biologically active surface display of peptides and the establishment of a solidly performing peptide microarray immunoassay are often troublesome in detail. The following protocols aim to provide facilitated access to the production of a robust peptide microarray platform and an optimized analytical processing of peptide microarrays in serological diagnosis. The functional surface display is accomplished by site-specific immobilization of peptide probes in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde-coated substrate for microstructured surface immobilization of the probe complexes. The serological assay is based on the specific capture of primary antibodies in a sandwich complex between the surface-immobilized peptides and a fluorescently labelled secondary antibody. A proposal for the diagnostic evaluation of fluorescence data obtained with the peptide microarray is also made.