Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. A large outbreak of CHIKV disease occurred in 2005 in the Indian Ocean Islands. Many cases have been imported in European countries. Laboratory confirmation of suspected cases is mandatory for control measures during an outbreak. We report a novel, real-time, reverse transcription-polymerase chain reaction (RT-PCR) for the nonstructural protein 1 region that can quantify a wide range of viral RNA concentrations. This assay was validated by in vitro experiments in which interferon-alpha, a well-known virus inhibitor, showed a dose-dependent inhibition of virus replication on Vero cells that was assessed by viral infectivity and viral RNA production. This new real-time RT-PCR was used to measure viral load in serum samples from cases recently imported to Italy, and may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication in patients.