Respiratory syncytial virus (RSV) and Influenza virus are the most common pathogen for causing severe upper respiratory infection in all age groups. A multiplex reverse transcription polymerase chain reaction (RT-PCR) has been developed to detect and subtype influenza A (H3N2 and H1N1), B virus and RSV simultaneously in one tube reaction. Amplification with primers derived from conserved sequences within the nucleocapsid for RSV and hemagglutinin subunit for Influenza A (H3N2 and H1N1) and B viruses yielded a 384 bp, a 300 bp, a 236 bp and a 151 bp, respectively. Assay specificity was confirmed by pulse field gel electrophoresis and autosequencing method. Assay sensitivity was 3 PFU/ml of RSV, 22 PFU/ml, 45 PFU/ml of Influenza type A (H3N2 and H1N1) and 6.6 PFU/ml of Influenza B virus by plaque assay. A rapid and sensitive detection method of a one-tube with multiplex RT-PCR capable of identifying more than one viral template as well as synchronizing reverse transcription and PCR had the potential to produce considerable savings of time and cost effectiveness in the diagnostic laboratory.