This work introduces a novel analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of type 1 diabetes. There is a clinical need for a rapid and automated assay for GAD65 autoantibodies. Therefore, this method was designed to exploit the advantages of bead injection (BI) analysis for enzyme-linked immunosorbent assays (ELISA). BI ELISA is a microscale technique that uses enzyme labeled secondary antibodies to detect the capture of target antibodies on immobilized antigen in the flow cell of the lab-on-valve (LOV) manifold. A detection limit of 20 ng mL(-1) of GAD65 monoclonal antibody 144 compares favorably with the sensitivity and precision of a standard ELISA currently employed to detect GAD65 autoantibodies. Compared to the standard ELISA protocol, BI ELISA offers a significantly reduced assay time and complete automation of solution handling and detection.