Circulating antibodies to glutamic acid decarboxylase (GADab) are a major indicator for autoimmune destruction of pancreatic islet cells (type 1 diabetes). Previously reported detection of GADab in oral fluid, however, was assayed by enzyme-linked immunosorbent assay (ELISA) with low diagnostic sensitivity and high non-specific binding. We re-assessed oral fluid GADab detection using a different sampling technique and a more robust assay. Type 1 diabetic subjects (n = 32; mean age +/- SD: 13.9 +/- 3.7 years) provided Orasure oral fluid and venous blood samples. Orasure collections were assayed for total immunoglobulin G (IgG), then concentrated to 1/10 of their volume using mini-centrifugal protein concentrators. All samples were assayed by a GAD65 antibody radio-immunoprecipitation method. Oral fluid antibodies were detected ( > 99th percentile of radio-binding (%counts per min (%cpm)) for seronegatives) in 10/16 seropositive subjects, with %cpm (median: 6.4%; range: 4.6-25.8) significantly greater (P < 0.001) than for seronegatives (median: 4.7%; range: 3.4-5.7). A highly significant correlation (Spearman's rho: 0.85; P<0.001) was demonstrated between %cpm of concentrates and respective serum titres for seropositive diabetics. Median IgG concentration of Orasure collections was 22.8 mg/l (range: 9.4-168.0). GADab recovery from Orasure collectors was estimated at 90%. This is the first confirmatory detection of diabetes-specific autoimmune markers in oral fluid. Acceptable correlation between concentrated oral fluid radio-binding and serum titre was achieved. Improved antibody recovery and assay re-optimisation could provide a basis for more extensive studies that may lead to an alternative non-invasive screening method for pre-clinical autoimmune diabetes.