The first application of nanocrystal (NC)-encoded microbeads to clinical proteomics is demonstrated by multiplexed detection of circulating autoantibodies, markers of systemic sclerosis. Two-color complexes, consisting of NC-encoded, antigen-covered beads, anti-antigen antibody or clinical serum samples, and dye-tagged detecting antibodies, were observed using flow cytometry assays and on the surface of single beads. The results of flow cytometry assays correlated with the ELISA technique and provided clear discrimination between the sera samples of healthy donors and patients with autoimmune disease. Microbead fluorescence signals exhibited narrow distribution regardless of their surface antigen staining, without the need of any fluorescence compensation-a parameter determining the limit of sensitivity of flow cytometry assays. In single bead measurements, less than 30 dye-labeled antibodies interacting with the topoI-specific antibodies at the surface of a bead have been detected by the emission of dye excited through the FRET from NCs. In this format, the antibody-bead interaction reaction turns specifically the fluorescence signal from dye label off and on, additionally increasing autoantibody detection sensitivity.