Abstract Background. Although it is the best method to detect early therapeutic failure, viral load (VL) monitoring is still not widely available in many resource-limited settings because of difficulties in specimen transfer, personnel shortage, and insufficient laboratory infrastructures. Dried blood spot (DBS) use, which was introduced in the latest World Health Organization recommendations, can overcome these difficulties. This evaluation aimed at validating VL measurement in DBS, in a laboratory without previous DBS experience and in routine testing conditions. Methods. Human immunodeficiency virus (HIV)-infected adults were observed in a HIV care site in Hanoi, and each patient provided 2 DBS cards with whole blood spots and 2 plasma samples. Viral load was measured in DBS and in plasma using the COBAS Ampliprep/TaqMan and the Abbott RealTime assays. To correctly identify those with VL ≥ 1000 copies/mL, sensitivity and specificity were estimated. Results. A total of 198 patients were enrolled. With the Roche technique, 51 plasma VL were ≥1000 copies/mL; among these, 28 presented a VL in DBS that was also ≥1000 copies/mL (sensitivity, 54.9; 95% confidence interval [CI], 40.3–68.9). On the other hand, all plasma VL < 1000 copies/mL were also <1000 copies/mL in DBS (specificity, 100; 95% CI, 97.5–100). With the Abbott technique, 45 plasma VL were ≥1000 copies/mL; among these, 42 VL in DBS were also ≥1000 copies/mL (sensitivity, 93.3%; 95% CI, 81.7–98.6); specificity was 94.8 (95% CI, 90.0–97.7). Conclusions. The Abbott RealTime polymerase chain reaction assay provided adequate VL results in DBS, thus allowing DBS use for VL monitoring.