Adequate adherence to anti-retroviral therapy is required to achieve viral suppression and desirable treatment outcomes among HIV patients. The aim of this study was to examine the associations between adherence and severity of substance use as well as adherence and severity of depressive symptoms among Iranian HIV patients. In a prospective study, HIV patients with current substance use were assessed for adherence level via self report and pill count methods, severity of depressive symptoms (Beck Depression Inventory- II) and substance use (Addiction Severity Index) during a three months follow up after initiating antiretroviral therapy. The adherence level, severity of depressive symptoms and substance use were assessed one month, two months and three months after initiation of anti-retroviral therapy. Addiction Severity Index (ASI) composite scores were calculated for each domain and the associations between ASI domains and adherence as well as severity of depressive symptoms and adherence were assessed. Twenty six HIV patients with current substance use disorder completed the study. At the end of the first month, adherence to therapy via pill count and self-report were 80%±31.9% and 85.12%±32%, respectively. At the end of the second month, adherence to therapy via pill count and self report were 87%±32% and 93.94%±23% respectively. At the end of the third month, the measured adherence via pill count and self report were 85%±33.7% and 90.1%±25.7% respectively. Adherence was higher among married patients and those who used reminder systems. Composite scores of the medical status and psychiatric status were related to higher adherence after first month. Substance use was inversely associated with adherence at the second follow up (r=-0.4, p=0.04). Also, severity of depressive symptoms was not related to adherence level. The repeated measurement analysis showed a significant decrease in psychiatric status domain of the ASI composite score after three months of initiating therapy (p=0.02). Preventive measures should aim treatment of substance use among HIV patients in order to increase adherence level. Also, conducting psychological evaluations is necessary considering the high prevalence of depression among Iranian HIV patients.