FluMist is an intranasal influenza live vaccine containing two Influenza A strains (currently H1N1 and H3N2) and one B strain (Yamagata or Victoria lineage). Characterization of the vaccine requires determination of the median tissue culture infectious dose (TCID(50)) titer, serum antivirus neutralization titer and vaccine cold adapted/temperature sensitive (ca/ts) phenotype. Visual cytopathic effect (CPE) readings are used widely in viral assays, but these are subjective and labor intensive. In response to the need for an efficient, inexpensive and high-throughput assay, a 96-well microplate assay was developed that uses Alamar blue dye staining as a replacement for CPE observation in the determination of influenza virus infectious dose, serum antivirus neutralization titer and virus ca/ts phenotype. Relative operating characteristic curves verified that there was a clear distinction between the fluorescence readings of the Alamar blue stained CPE positive and CPE negative wells. Virus titer was determined by use of both Alamar blue staining and CPE-based TCID(50) assays for wild-type and FluMist influenza vaccine strains as well as a plasmid-rescued influenza FluMist A strain containing a H5N1 derived hemmaglutinin and neuramidinase. Correlation of the two assays was measured by regression analysis and resulted in R(2) values of 0.814 (Influenza A), 0.983 (Influenza B) and 1.000 (H5N1), respectively. Serum microneutralization as well as virus ca/ts phenotype assays also showed a high concordance between readings based on CPE observation and Alamar blue staining. The Alamar blue dye assay is user friendly, environmentally safe and sensitive. Also, it is adaptable to automation, which could provide a high-throughput platform for analysis of pre-clinical and clinical samples.