ABSTRACT Dried blood spot (DBS) may overestimate the viral RNA, mainly in patients with low viral load (VL), due to proviral DNA and intracellular RNA. The Burnett and HemaSpot provide integrated solutions for the collection, separation, and drying of plasma from whole blood. This study aims to evaluate the performance of both devices compared to plasma to identify antiretroviral therapy (ART) failure. The devices were separately evaluated in a cross-sectional design. Patients on ART were included for the studies (Burnett: 611, October 2019 to January 2020) and (HemaSpot: 620, November 2020 to April 2021). VL was tested using CAP/CTM96. The sensitivity and specificity of DBS, Burnett, and HemaSpot were determined, and plasma results were considered as a reference at a threshold of 1,000 copies/ml. For the Burnett study, 2,444 specimens, including plasma, DBS, venous Burnett (vBurnett), and capillary Burnett (cBurnett), were collected. Sensitivity of DBS, vBurnett, and cBurnett was 97.4%, 98.3%, and 97.5%, respectively, whereas specificity was 86.8% for DBS, 96.9% for vBurnett, and 93.9% for cBurnett. For the HemaSpot study, 1,860 specimens were collected, including plasma, DBS, and vHemaSpot. Sensitivity of DBS and vHemaSpot was 95.0% and 91.3%, respectively, whereas specificity was 86.9% for DBS and 94.5% for vHemaSpot. The misclassification rate was more prominent in DBS (4.8%) and HemaSpot (8.4%) but lower in vBurnett (2.0%) and cBurnett (3.2%). The Burnett showed better performance than DBS, whereas HemaSpot showed poorer performance than DBS. Nevertheless, both Burnett and HemaSpot have high rate of non-reportable results. In the current format, neither of the two devices is feasible for VL scale-up in resource-limited settings. IMPORTANCE Burnett and HemaSpot are two novel technologies that allow whole blood collection and plasma separation and stabilization at room temperature without the need of additional equipment. Hence, these devices are potential alternatives to fresh plasma as a suitable specimen for viral load scale-up to monitor antiretroviral therapy in resource-limited settings