HIV replication in humans proceeds with substantial viral RNA levels in plasma. Antiretroviral therapy results in suppression but not eradication of HIV infection. Continuous therapy is essential for durable clinical responses. Discontinuing antiretroviral therapy results in prompt rebound in viremia. The source of HIV during suppressive therapy and mechanisms of persistence remain uncertain. Sensitive assays for HIV have been useful in quantifying viremia in response to antiretroviral therapy and in experimental studies of drug intensification, drug simplification, and potential anatomic sanctuary site investigations. As clinical eradication strategies move forward, robust, sensitive quantitative assays for HIV at low levels represent essential laboratory support modalities. Here we describe in detail an assay for HIV-1 RNA with single-copy sensitivity.