Current methods for in vitro diagnosis of autoimmune diseases (ADs) are mainly based on the detection of circulating autoantibodies using specific AD autoantigens. A high-quality recombinant autoantigen with epitopes resembling its natural form in the human body is the key to sensitive, specific, and rapid diagnosis. In this webinar we describe the use of the Gibco™ Bac-to-Bac™ Baculovirus Expression System to express 22 recombinant autoantigens in Spodoptera frugiperda 9 (Sf9) cells, with folding and posttranslational modifications matching those of the natural autoantigens in vivo. The Bac-to-Bac system allowed us to generate recombinant baculoviruses expressing the target genes via transposition within 48 hours. Using Gibco™ Sf-900™ II SFM, we were able to propagate Sf9 cells to a density of approximately 6–8 x 106 cells/mL in a suspension culture and over 1–2 x 107 cells/mL during fermentation. These tools significantly reduce the time and labor costs needed for virus generation, amplification, and protein production, and allow high-throughput production of autoantigens. Using these 22 high-quality recombinant autoantigens, we have successfully developed a membrane-based chip for simultaneous screening of 7 major ADs that were validated in over 200 clinical samples. In addition to demonstrating greater than 95% sensitivity, the new assay procedure is very rapid (only taking 30 minutes).