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Most HIV-infected subjects on antiretroviral therapy (ART) in resource-limited settings do not undergo virologic monitoring. There is an urgent need for cheap, accessible HIV RNA assays for early diagnosis of virologic failure. We investigated filter paper transfer (FPT) of whole blood and plasma as an alternative to standard plasma-based assays for virologic monitoring in Uganda.Whole blood (n = 306) and plasma (n = 218) from 402 subjects established on ART were spotted onto filter paper and transported to Europe for HIV RNA extraction and quantification. These results were compared to a gold standard plasma assay in Kampala.Of 402 ART-treated subjects, 39 (9.7%) had viremia detectable (>500 copies/mL) by local methods. Plasma FPT showed excellent agreement with gold standard, whereas whole blood yielded a large number of false-positive viral loads.This is the first study to investigate the use of FPT in ART-treated subjects and demonstrates that it may provide a practical, reliable method for virologic monitoring in resource-poor settings. Plasma FPT was accurate but requires centrifuge; whole blood produced a high number of false-positive results, but these were low-level. Whole blood may be sufficiently accurate if higher HIV RNA cut-offs were used to define virologic failure.