Objective To evaluate resource allocation and costs associated with delivery of human immunodeficiency virus (HIV) services in Uganda and the United Republic of Tanzania. Methods We used time-driven activity-based costing to determine the resources consumed and costs of providing five HIV services in Uganda and the United Republic of Tanzania: antiretroviral therapy (ART); HIV testing and counselling; prevention of mother-to-child transmission; voluntary male medical circumcision; and pre-exposure prophylaxis. Findings Country-based teams undertook time-driven activity-based costing with 1119 adults in Uganda and 886 adults in the United Republic of Tanzania. In Uganda, service delivery costs ranged from 8.18 United States dollars (US$) per visit for HIV testing and counselling to US$ 43.43 for ART (for clients in whom HIV was suppressed). In the United Republic of Tanzania, these costs ranged from US$ 3.67 per visit for HIV testing and counselling to US$ 28.00 for voluntary male medical circumcision. In both countries, consumables were the main cost driver, accounting for more than 60% of expenditure. Process maps showed that in both countries, registration, measurement of vital signs, consultation and medication dispensing were the steps that occurred most frequently for ART clients. Conclusion Establishing a rigorous, longitudinal system for tracking investments in HIV services that includes thousands of clients and numerous facilities is achievable in different settings with a high HIV burden. Consistent engagement of implementation partners and standardized training and data collection instruments proved essential for the success of these exercise