The use of nucleic acid detection for HIV type 1 (HIV-1) detection is strongly recommended in infants<18 months of age, in whom serology is unreliable. This study evaluated the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test v2.0 (TaqMan HIV-1 Qual Test, v2.0), a dual-target total nucleic acid real-time PCR assay. The limit of detection (LOD) of the new test in plasma and dried blood spots (DBS) was determined with the 2nd International HIV-1 RNAWHOstandard. The specificity of the assay was tested with EDTA plasma (n1,301) and DBS from HIV-negative adults (n1,000). The sensitivity was determined using HIV-1-positive samples (n169 adult EDTA plasma, n172 adult DBS, and n100 infant DBS) that included group M, subtypes A to H, CRF01-AE, CRF02-AG, and groups O and N. All positive specimens and a subset of the negative specimens were also tested with the Abbott RealTime HIV-1 Qual assay (RealTime). The LOD of the TaqMan assay was 20 copies/ml in plasma and 300 copies/ml in DBS, with specificities of 99.8% in plasma and 99.9% in DBS. The TaqMan assay results were 100% concordant with RealTime results in EDTA plasma samples and in 100 HIV-1-negative adult DBS. Among 172 HIV-1-positive DBS from adults, the TaqMan assay showed positive results for all DBS while RealTime missed five DBS with low target concentrations. Infant DBS results were 100% concordant. The improved sensitivity of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test, v2.0, compared to current commercially available assays may enable earlier diagnosis and treatment in adults and infants. The dual-target test may ensure HIV-1 detection even if a mutation is present in one of the two target regions. The DBS sample matrix facilitates virological testing in remote areas.