Introduction and Aims. The acceptability of testing methods and procedures has implications for uptake of blood-borne virus screening in sentinel samples of injecting drug users (IDUs) likely to participate in surveillance. The aim of the current study was to determine the acceptability of three methods of hepatitis C virus (HCV) testing among injecting drug users (IDUs): oral fluid, capillary blood and venous blood sampling. Design and Methods. A cross-sectional survey of IDUs was conducted in inner-city Sydney in 2005 for a laboratory validation study of HCV antibody testing. Participants were tested using the three different specimen collection methods and asked about the acceptability of each method and a particular preference documented. Results. Two-hundred and twenty-nine IDUs participated in the study. Before and after specimen collection, the acceptability of all three collection methods for HCV testing was high (>85%). Oral fluid remained the preferred method after sample collection, with females (65%) significantly more likely than males (49%) to report a preference (unadjusted odds ratio 2.0; 95% confidence interval 1.1–3.5, p = 0.03) for that method. Discussion and Conclusions. Findings suggest that oral fluid testing is an acceptable and preferred alternative for HCV testing among IDUs. However, concerns reported by participants in the study indicate that information and education regarding the nature and diagnostic value of oral fluid testing is necessary prior to its implementation for surveillance purposes among this population.