The development of autoantibodies is observed in autoimmune disorders and numerous cancers. Consequently, autoantibodies form the basis of potential diagnostic and prognostic assays, as well as approaches for monitoring disease progression and treatment response. The effective use of autoantigen biomarkers for these applications, however, is contingent upon the identification of not one but multiple biomarkers. This is a consequence of the observation that the development of autoantibodies to any given protein is typically seen only in a fraction of patients. We have previously demonstrated the utility of functional protein microarrays containing thousands of different human proteins (ProtoArrays) for discovering novel autoimmune biomarkers in serum and plasma. Here, we describe a protocol for detecting autoantibodies in urine.