INTRODUCTION: The use of antigen rapid tests (Ag-RDTs) for self-testing is an important element of the COVID-19 control strategy and has been widely supported. However, scale-up of self-testing for COVID-19 in sub-Saharan Africa is still insufficient and there is limited evidence on the acceptability of self-testing and agreement between Ag-RDT self-testing and Ag-RDT testing by professional users. A joint collaboration (Botnar Research Centre for Child Health-European & Developing countries Clinical Trials Partnership)was established between Lesotho and Zambia to address these gaps in relation to Ag-RDT self-testing and contribute to increasing its use in the region. METHODS: A cross-sectional study was conducted with qualitative and quantitative data analysis. Firstly, 14 in-depth cognitive interviews (5 in Zambia and 9 in Lesotho) were performed to assess the participants' understanding of the instructions for use (IFU) for self-testing. In a second step, evaluation of test agreement between Ag-RDT self-testing and Ag-RDT testing by professional user using SD Biosensor STANDARD Q COVID-19 Ag-RDT was performed. In Zambia, usability and acceptability of self-testing were also assessed. RESULTS: Cognitive interviews in Lesotho and Zambia showed overall good understanding of IFU. In Zambia, acceptability of self-testing was high, though some participants had difficulties in conducting certain steps in the IFU correctly. Agreement between Ag-RDT self-test and Ag-RDT by professional users in Lesotho (428 participants) and Zambia (1136 participants) was high, 97.3% (403/414, 95% CI: 95.3-98.7) and 99.8% (1116/1118, 95% CI: 99.4-100) respectively. CONCLUSION: Findings from this study support the use of Ag-RDT self-testing within COVID-19 control strategies in sub-Saharan Africa, contributing to increase the testing capacity and access in hard-to reach settings.