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Background. Quantitative human immunodeficiency virus (HIV) RNA load testing surpasses CD4 cell count and clinical monitoring in detecting antiretroviral therapy (ART) failure; however, its cost can be prohibitive. Recently, the use of pooling strategies with a clinically appropriate viral load threshold was shown to be accurate and efficient for monitoring when the prevalence of virologic failure is low. Methods. We used laboratory request form information to identify specimens with a low pretest probability of virologic failure. Patients aged⩾15 years who were receiving first-line ART had individual viral load results available were eligible. Blood plasma, dried blood spots, and dried plasma spots were evaluated. Two pooling strategies were compared: minipools of 5 samples and a 10 ×10 matrix platform (liquid plasma specimens only). A deconvolution algorithm was used to identify specimens(s) with detectable viral loads. Results. The virologic failure rate in the study sample was <10%. Specimens included were liquid plasma specimens tested in minipools(n 5 400), of which 300 were available for testing by matrix, and specimens tested with minipools only: dried blood spots (n = 100) and dried plasma spots (n = 185). Pooling methods resulted in 30.5%-60% fewer HIV RNA tests required to screen the study sample. For plasma pooling, the matrix strategy had the better efficiency, but minipools of 5 dried blood spotshad the best efficiency overall and were accurate at a >95% negative predictive value with minimal technical requirements. Conclusions. In resource-constrained settings, a combination of preselection of patients with low pretest probability of virologic failure and pooled testing can reduce the cost of virologic monitoring without compromising accuracy. [ABSTRACT FROM AUTHOR]