Identification of B-cell epitopes is important for the use of antibodies as therapeutic agents, the design of epitope-based vaccines against infectious diseases, and immunological assays based on peptide antibodies. A large number of methods are available for epitope mapping, but many of them require specialized laboratories and are expensive. In this chapter, we describe a high-throughput approach for epitope mapping of peptide antibodies by use of a library of soluble, overlapping, biotinylated peptides. As example, we present characterization of monoclonal and polyclonal antibodies specific for peptides of Mycobacterium tuberculosis acyl carrier protein AcpM and the Chlamydia trachomatis chaperone Ct043 by ELISA.