Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized 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 glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4C1 was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406-411) and A10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases.