Accurate treatment knowledge is required for patients to successfully manage complex medical conditions. Existing HIV knowledge scales focus on disease transmission and risk factors. This is the first study to develop and validate a scale to measure HIV treatment knowledge about complex treatment issues such as adherence, side-effects and drug resistance. A total of 346 participants were recruited into this cross-sectional study. Participants included HIV-positive patients (n=130), HIV-hepatitis C co-infected patients (n=22), hepatitis C patients, (n=78), community healthcare providers (n=35) and college students (n=81). Participants completed the proposed HIV Treatment Knowledge Scale and a validated measure of general knowledge about HIV transmission and risk factors. Two-week test-retest data were collected. Results demonstrated that the HIV Treatment Knowledge Scale was significantly correlated with general HIV knowledge across all samples. Among HIV-positive patients, the HIV Treatment Knowledge Scale was positively associated with time since HIV diagnosis. HAART-experienced patients had significantly higher treatment knowledge than HAART-naïve patients. HIV-positive patients scored significantly higher than hepatitis C patients and college students on HIV treatment knowledge. Test-retest reliability (r=0.83) and internal consistency (reliability coefficient=0.90) were both satisfactory. The HIV Treatment Knowledge Scale is a novel, easy-to-administer measure demonstrating high levels of validity and reliability. It has important applications as a clinical teaching tool with patients and healthcare workers and it could be used as an outcome indicator in HIV educational intervention studies.