Although conventionally the detection of HCV antibodies is carried out on serum, the collection of oral fluid is non-invasive, safe and cost effective. In this study, the efficacy of the detection of HCV antibodies in oral fluid was assessed. 73 anti-HCV positive and 73 anti-HCV negative paired serum/oral fluid samples, drawn from patients visiting a Belgian academic hospital, were tested using the modified Ortho HCV 3.0 and LIA confirmation assay. Performing the test on oral fluid with the modified protocol, 61/73 anti-HCV positive samples were tested positive, while 73/73 anti-HCV negative samples were tested negative, giving a sensitivity and specificity of 83.6% (95% CI: 72.7-90.9%) and 100.0% (95% CI: 93.8-100.0%), respectively. Comparing S/CO of concordantly positive and negative samples, the cut-off point was lowered by 30% resulting in a sensitivity of 89.0% (95% CI: 79.0-94.8%) while the specificity remained 100.0% (95% CI: 93.8-100.0%). The confirmation assay was carried out as described by the manufacturer, diluting the oral fluid 1:10. Testing paired samples gave a concordance of 85.6% (125/146), yielding no more accurate results. These findings suggested that the modified ELISA method for anti-HCV detection in oral fluid can be used for epidemiological surveys.