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Abstract: Background: Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, like culture and antigen detection. However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR''s is limited for most published respiratory multiplex real time PCR assays. Objectives: Development and extensive analysis of an internally controlled multiplex real time rt-PCR for detection of respiratory viruses. Study design: The assay was validated in comparison to single-target PCR''s using plasmid targets and prospectively collected nasopharyngeal aspirates. Results: Using plasmid targets the multiplex format was found to be as least as sensitive and specific as the single-target PCR and no competition was observed when different targets were present at different amounts in one tube. Clinical validation showed high concordance for all viruses tested except for samples with low levels of enterovirus. Conclusion: This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus. [Copyright &y& Elsevier]