Little is known about how CD4 and viral load testing have evolved followingimplementation of universal test and treat (UTT) in African settings. Wereviewed World Health Organization (WHO) guidance from 2013 to 2018,and compared it against national HIV policies in Malawi, Tanzania andSouth Africa. Three surveys rounds were conducted in 2013, 2016 and2017–2018 in 33 health facilities across the three settings to assessimplementation of national policies on the use of biological markers.Qualitative interviews were conducted with 26 HIV policymakers orprogramme managers, 21 providers and 66 people living with HIV toexplore understandings and experiences of these tests. Various factorsinfluenced adoption and implementation of WHO guidance, includinghistorical policies on CD4 counts, governance issues, supply chainchallenges and funding mechanisms. Facility-level practices relating to theuse of these tests often diverged from national policies. Patients andproviders valued both tests, but did not always understand their roles. Inaddition to continued support for scaling-up viral load testing, renewedfocus should be placed on the ongoing value of point-of-care CD4 tests inthe UTT era, including its role in assessing disease progression andinforming clinical management of cases to reduce HIV-related mortality.