Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.