The preliminary positive effects of B cell depletion therapy in multiple sclerosis (MS) have renewed interest in a potential role of B cells and autoantibodies in the MS disease process. Regardless of a possible pathogenic role of the humoral immune response in MS, the analysis of autoantibodies as disease markers is valuable. Despite intense research, there is no known MS-associated antibody specificity that can individually discriminate between MS patients and controls. Due to the overlap in autoantibody profiles in autoimmune diseases, and due to the complexity of MS, multiplex autoantibody profiling approaches are needed to generate a panel of MS-associated autoantibodies with high combined sensitivity and specificity for MS. In recent years, several multiplexing approaches have been applied in MS autoantibody profiling with promising results regarding the generation of a so-called MS-specific autoantibody fingerprint. We also recently applied a high-throughput autoantibody profiling technique for MS cerebrospinal fluid resulting in the identification of a novel panel of 8 antigenic targets with 45% sensitivity and 86% specificity for the disease. Identification of MS-specific autoantibody specificities is important for the development of diagnostic and prognostic markers for MS. Moreover, it can provide more knowledge regarding underlying MS disease processes and novel therapeutic targets.