In order to detect and identify dengue serotypes in serum samples, we developed a single-step quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay (referred to as Q-PCR). Sets of primers were selected from the capsid region of the viral genome. Dengue serotypes 1/3 and 2/4 were detected in two separate duplex amplification reactions using specific primers and fluorogenic TaqMan probes. Results obtained with this Q-PCR and the classical nested RT-PCR (N-PCR) assays were compared using a panel of 97 representative human sera collected from patients in Bangkok, Thailand. It is shown that the Q-PCR is a rapid, sensitive and reproducible tool for the detection and quantitation of the four dengue serotypes in clinical samples, and therefore of great interest for diagnostic use or for large cohort studies.