INTRODUCTION: Cross-sectional incidence testing is used to estimate population-level HIV incidence and measure the impact of prevention interventions. There are limited data evaluating the accuracy of estimates in settings where antiretroviral therapy coverage and levels of viral suppression are high. Understanding cross-sectional incidence estimates in these settings is important as viral suppression can lead to false recent test results. We compared the accuracy of multi-assay algorithms (MAA) for incidence estimation to that observed in the community-randomized HPTN 071 (PopART) trial, where the majority of participants with HIV infection were virally suppressed. METHODS: HIV incidence was assessed during the second year of the study, and included only individuals who were tested for HIV at visits 1 and 2 years after the start of the study (2016-2017). Incidence estimates from three MAAs were compared to the observed incidence between years 1 and 2 (MAA-C: LAg-Avidity <2.8 ODn + BioRad Avidity Index <95% + VL >400 copies/ml; LAg+VL MAA: LAg-Avidity <1.5 ODn + VL >1000 copies/ml; Rapid+VL MAA: Asanté recent rapid result + VL >1000 copies/ml). The mean duration of recent infection (MDRI) used for the three MAAs was 248, 130 and 180 days, respectively. RESULTS AND DISCUSSION: The study consisted of: 15,845 HIV-negative individuals; 4406 HIV positive at both visits; and 221 who seroconverted between visits. Viral load (VL) data were available for all HIV-positive participants at the 2-year visit. Sixty four (29%) of the seroconverters and 3227 (72%) prevelant positive participants were virally supressed (<400 copies/ml). Observed HIV incidence was 1.34% (95% CI: 1.17-1.53). Estimates of incidence were similar to observed incidence for MAA-C, 1.26% (95% CI: 1.02-1.51) and the LAg+VL MAA, 1.29 (95% CI: 0.97-1.62). Incidence estimated by the Rapid+VL MAA was significantly lower than observed incidence (0.92%, 95% CI: 0.69-1.15, p<0.01). CONCLUSIONS: MAA-C and the LAg+VL MAA provided accurate point estimates of incidence in this cohort with high levels of viral suppression. The Rapid+VL significantly underestimated incidence, suggesting that the MDRI recommended by the manufacturer is too long or the assay is not accurately detecting enough recent infections.