The application of flow cytometry measurements initially analyzed hematopoietic and other mammalian cell types. Today it has the ability to analyze whole cells and their constituents, as well as subcellular components. In addition, through improvements in detection sensitivity, flow cytometry can now analyze individual molecules. There are now several applications of the basic flow cytometry to high-throughput research emerging in the biodefense arena. This chapter examined two examples of flow cytometric measurements that have application in the biodefense field, DNA fragment size analysis and microsphere-based assays for multiplexed minisequencing and toxin detection. DNA fragment sizing technology has the capability of recognizing and identifying both bacterial species and strains and is useful in forensic applications for determining the source of a biothreat agent, identifying and tracking outbreaks, and to confirm the identity of biothreat agents by other means. The applications of microsphere-based assays include the measurement of enzyme activity, immunoassays, and single nucleotide polymorphism (SNP) detection. When biothreat organisms are sequenced the microsphere-based assay technique is used to determine agent species and strains. The microsphere-based cholera toxin assay uses the fluorescence resonant energy transfer principle resulting in a fluorescence emission spectrum from the bead that is characteristic of the donor molecule. Future developments include a new technology enabling multiplexing of assays. These developments can enable high-throughput sample preparation and analysis taking advantage of the flow cytometer's ability to analyze large numbers of individual particles. References