Array-based biosensor technology offers the user the ability to detect and quantify multiple targets in multiple samples simultaneously (Analytical Sciences 23:5–10, 2007). The NRL Array Biosensor has been developed with the aim of creating a system for sensitive, rapid, on-site screening for multiple targets of interest. This system is fluorescence-based, using evanescent illumination of a waveguide, and has demonstrated the use of both sandwich and competitive immunoassays for the detection of both high and low molecular weight targets, respectively. The current portable, automated system has demonstrated detection of a wide variety of analytes ranging from simple chemical compounds to entire bacterial cells, with applications in food safety, disease diagnosis, homeland security and environmental monitoring.