Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP(Sc), a misfolded isoform of the normal host-encoded prion protein PrP(C). Immunodetection of PrP(Sc) is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP(Sc). This assay has PrP(Sc) detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.