In this study, we used a nucleic acid based real-time PCR assay to detect and quantify plasma HIV-1 copy numbers on samples from 56 HIV-1 infected patients utilizing the DBS format. The assay results were comparable and correlated well with commercially available viral load assays (Siemens bDNA, Spearman r = 0.89 and Roche Ultrasensitive Amplicor, Spearman r = 0.91). In the patient cohort analyzed, the assay successfully detected all positive samples. The calculated specificity using known negative samples was 100%. The estimated 95% detection threshold was 136 copies and the dynamic range of the assay was 5 log 10 . Finally, the assay successfully detected four major subtypes and 2 CRF of HIV-1.