One of the first applications of peptide arrays in general and those prepared by SPOT synthesis in particular was the mapping of antibody epitopes. In this article the diverse applications in this field are described. Different types of peptide libraries such as peptide scans, substitution analyses, truncation and deletion libraries as well as combinatorial and randomly generated libraries are briefly covered. Furthermore, their applications for antibody epitope mapping are described. These are: (i) identification of antigenic determinants within protein sequences for monoclonal and polyclonal antibodies, (ii) identification of key residues for binding, mapping of linear and discontinuous binding sites, (iii) paratope mapping, (iv) identification of mimotopes, and finally (v) the profiling of complex (auto)antibody signatures in biological fluids such as human or mice sera to identify novel biomarkers for e.g. cancer, allergy, infectious and autoimmune diseases.