Current assays for quantification of HIV-1 virions rely on real-time reverse transcriptase (RT)-PCR detection of conserved regions of HIV-1 RNA and can be limited by detection of contaminating viral or plasmid DNA. We developed a novel RT-PCR assay using a reverse primer that hybridizes with the poly(A) tail of HIV-1 mRNAs, anchored by conserved viral nucleotides at the most distal region of the transcript. This assay can detect and quantify HIV-1 RNA with high specificity and sensitivity.