Plasma viral RNA load is a key parameter in disease progression of lentiviral infections. To measure simian immunodeficiency virus (SIV) RNA loads, we have established a quantitative one-tube assay based on TaqMan chemistry. This real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has advantages compared with previous methods, such as higher sensitivity, shorter time consumption, and low risk of cross-contamination. The sensitivity of the assay was optimized by comparing different enzyme systems. The one-enzyme protocol using rTth DNA polymerase was superior to two assays employing two enzymes. It detects 100% of the samples containing four copies of RNA transcript and allows quantification of viral RNA loads over an 8-log unit dynamic range. As few as 50 copies per milliliter of plasma can be detected within RNA extracted from 140 μl of plasma. This is especially relevant in studies employing neonatal macaques, from which only small volumes of blood can be sampled, and in studies in which low viral RNA loads are expected. Because of the use of rTth DNA polymerase, DNA contamination can be avoided by carryover prevention with uracil N-glycosylase (UNG). We demonstrate that for optimization of real-time PCR sensitivity, not only concentrations of different reagents but also different enzyme systems must be evaluated. Our assay facilitates and enhances the quantification of plasma RNA loads, a critical parameter for many studies, including evaluations of vaccine candidates or antiviral regimens.