Human immunodeficiency virus type 2 (HIV-2) is distantly related to the more widespread HIV-1. Although HIV-2 infection is rare in the U.S., cases are concentrated in the Northeast. No FDA-approved HIV-2 viral load assays exist. A clinically validated laboratory-developed assay is currently available in the U.S., however it is not currently approved for use on New York State patients.To develop a sensitive viral load assay to quantify HIV-2 RNA in plasma and to validate it for clinical use.The real-time RT-PCR assay simultaneously amplifies HIV-2 and a whole virus internal control, added during the lysis step. Two extraction volumes can be used. Results are reported in HIV-2 RNA International Units (IU).The assay has a limit of detection of 7IU/mL and a lower limit of quantification of 29IU/mL. The assay detects multiple strains of HIV-2 group A and B and generates reproducible results. Samples exchanged with a comparator laboratory produced similar viral load results, with 74% of positives differing by <0.5log 10 IU/mL. To date, we have tested 52 clinical specimens from 25 individuals. Twenty-eight (54%) specimens had measurable HIV-2 viral loads (range: 1.63–5.14log 10 IU/mL), 10 (19%) were positive but not quantifiable, and 14 were negative. HIV-2 RNA was detected in at least one specimen from 19 of 25 (76%) individuals tested.We developed a sensitive and accurate HIV-2 viral load assay. Validation data indicate the assay is suitable for clinical use and its availability in New York State will improve clinical monitoring of HIV-2 infected patients.