The availability of rapid, sensitive and cost-effective diagnostic methods is paramount to the success of a comprehensive national health security system in the USA. The national networks that were established to safeguard US infrastructures (e.g., public health, livestock, agriculture and water supply) have developed sufficient capability and capacity for monitoring. However, additional advanced methods will be required to maintain operational readiness. Currently available methods, although sensitive and specific, are generally costly and not amenable to high-throughput analyses. Critical to the success of biothreat surveillance is the ability to screen for and detect multiple agents rapidly in a single reaction and with minimal sample processing. This review will examine currently available diagnostic platforms (i.e., PCR-, immuno- and array-based) and biosensors that can detect multiple biothreat analytes in a single reaction (i.e., multiplex assays). The maturity, benefits and limitations of each platform will be described and a prospective view, from current to future state of the art, will be proposed.