Transmissible spongiform encephalopathies, or prion diseases, are a group of fatal neurodegenerative illnesses that affect humans and other mammalian species. All forms of prion disease are associated with misfolding of the host-encoded prion protein, PrP^C , into one of several pathogenic isoforms, collectively termed PrP^Sc . The current bioassays for prions typically involve intracerebral or peripheral inoculation of test material into an experimental host and subsequent euthanasia when clinical signs of terminal prion disease become evident. Consequently, bioassays of prion infectivity invertebrate species are cumbersome, time-consuming, expensive, and increasingly open to ethical dispute because the test animals are subjected to terminal neurodegenerative disease. Here, we discuss the development of a Drosophila-based prion bioassay, a highly sensitive and rapid invertebrate in vivo assay that efficiently identifies mammalian prions and permits the reduction and replacement of sentient experimental animals. This article provides an overview of the method ology involved in the model and discusses the experimental data that describe its viability and usefulness in place of more sentient species.