Multiple factors make adherence to antiretroviral therapy (ART) a complex process. This study aims to describe the barriers and facilitators to adherence for patients receiving first-line and second-line ART, identify different adherence strategies utilized and make recommendations for an improved adherence strategy. This mixed method parallel convergent study will be conducted in seven high volume public health facilities in Gauteng and one in Limpopo province in South Africa. The study consists of four phases; a retrospective secondary data analysis of a large cohort of patients on ART (using TIER.Net, an ART patient and data management system for recording and monitoring patients on ART and tuberculosis (TB)) from seven Johannesburg inner-city public health facilities (Gauteng province); a secondary data analysis of the Intensified Treatment Monitoring Accumulation (ITREMA) trial (a randomized control trial which ran from June 2015 to January 2019) conducted at the Ndlovu Medical Center (Limpopo province); in-depth interviews with people living with Human Immunodeficiency Virus (PLHIV) who are taking ART (in both urban and rural settings); and a systematic review of the impact of treatment adherence interventions for chronic conditions in sub-Saharan Africa. Data will be collected on demographics, socio-economic status, treatment support, retention in care status, disclosure, stigma, clinical markers (CD4 count and viral load (VL)), self-reported adherence information, intrapersonal, and interpersonal factors, community networks, and policy level factors. The systematic review will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) reporting and Population, Interventions, Comparisons and Outcomes (PICO) criteria. Analyses will involve tests of association (Chi-square and t-test), thematic analysis (deductive and inductive approaches) and network meta-analysis. Using an integrated multilevel socio-ecological framework this study will describe the factors associated with adherence for PLHIV who are taking first-line or second-line ART. Implementing evidence-based adherence approaches, when taken up, will improve patient's overall health outcomes. Our study results will provide guidance regarding context-specific intervention strategies to improve ART adherence.