An electric-field-driven assay for fluorescein-labeled staphylococcal enterotoxin B and cholera toxin B was developed on an active electronic microchip. An array of microlocations was transformed into an immunoassay array by electronically biasing electrodes at each microlocation to attract biotinylated capture antibodies. The electric field generated on the array directed the transport, concentration, and binding of biotinylated capture antibodies to streptavidin-coated microlocations. Subsequently, solutions of fluorescein-labeled staphylococcal enterotoxin B and fluorescein-labeled cholera toxin B were electronically addressed to the assay sites by an applied electric field. Each toxin was specifically bound to microlocations containing the appropriate capture antibody with little nonspecific binding to assay sites lacking the appropriate capture antibody. It was possible to detect both toxins from a mixture in a single electronic addressing step; detection was accomplished after a 1-min application of the electric field followed by washing. The ability to perform a rapid, electric field-mediated immunoassay for multiple analytes may provide an advantage over existing approaches.