HIV self-testing has emerged as a safe and effective approach to increase the access to and uptake of HIV testing and treatment, especially for key populations. Applying self-testing to hepatitis C virus (HCV) may also offer an additional way to address low coverage of HCV testing and to accelerate elimination efforts. To understand the potential for HCV self-testing (HCVST), an observational study was conducted to assess the acceptability and usability of the OraQuick® HCV Self-Test (prototype) among people who inject drugs (PWID) and men who have sex with men (MSM) in Thai Nguyen, a province in northern Vietnam. A total of 105 PWID and 104 MSM were eligible and agreed to participate in the study. Acceptability, defined as the proportion of participants among eligible subjects who agreed to participate in the study, was 92.9% in PWID and 98.6% in MSM. Compared to MSM, PWID were older (median age: 45 vs. 22 years; p < 0.0001) and had a lower education level (high school and college: 38.1% vs. 100%; p < 0.0001). HCVST usability was high among MSM with fewer observed mistakes, difficulties, or participants requiring assistance (33.7%, 28.8%, and 17.3%, respectively) compared to PWID (62.9%, 53.3%, and 66.7%, respectively; all p < 0.0001)). Inter-reader and inter-operator agreement were good in both groups (Kappa coefficient range: 0.61-0.99). However, the concordance between HCVST and study staff -read or performed HCV testing was lower among PWID than MSM (inter-reader concordance 88.6% vs. 99.0% and inter-operator concordance 81.9% vs. 99%). Overall, HCVST was highly acceptable with moderate to high usability among PWID and MSM in Thai Nguyen. Efforts to provide support and assistance may be needed to optimize performance, particularly for PWID populations and for those who are older and with lower literacy or education levels.