Multiple studies have examined the use of oral fluids in modified serum-based assays aiming to replace serum in antibody detection for hepatitis A. However, the reliable detection of HAV immunity in oral fluid requires an extremely sensitive assay; most immunoassays designed for serum antibody determination lack sufficient sensitivity for this purpose. Consequently, an "in-house" competitive enzyme immunoassay (EIA) designed specifically for use with oral samples collected using a ChemBio(®) device was developed to detect total anti-HAV antibodies (IgG and IgM). This system was compared to an in-house competitive EIA and a commercial EIA considered to be the "gold standard" using corresponding serum samples (n=225) to determine the accuracy of the assay and to evaluate the importance of the cutoff ratio for the detection of anti-HAV antibodies in oral fluids. When the median serum cutoff and the optimal oral fluid cutoff (ROC analysis) obtained from the in-house competitive EIA were compared, the oral fluid cutoff was found to be 28.8% higher than the serum cutoff. When different oral fluid cutoff values were compared, a reduction of about 17% was shown to be essential to increase test accuracy. At an oral fluid cutoff value of 0.351, sensitivity and specificity were higher, reaching 91.7% and 86.2% (p<0.001, AUROC=0.915), respectively. The convenience, accuracy and non-invasive nature of the developed method make it a useful alternative to serum-based assays for discriminating between HAV-immune and non-immune individuals.