World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can distinguish between DNA and RNA is used (e.g. bioMérieux EasyQ® or Abbott RealTime). There is a need for additional affordable technologies with the ability to discriminate between cell-free (plasma) and cell-associated nucleic acids.