To assess the utility of the RT-PCR/ESI-MS assay for surveillance of influenza virus in human populations, we analyzed 656 blinded clinical samples collected over a seven-year period (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) . The results were compared with conventional analysis of the same samples by virus culture/serology and real-time RT-PCR methods. Two hundred forty-three samples were influenza positive both by RT-PCR/ESI-MS and conventional assays. Ten samples were positive only by RT-PCR/ESI-MS while eight samples were positive only by culture/real-time RT-PCR, corresponding to approximately 97% sensitivity and 98% specificity. Of the influenza-positive samples, RT-PCR/ESI-MS analysis identified 186 as influenza A virus and 67 as influenza B virus, in complete agreement with conventional typing methods.