Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications . IMPORTANCE The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.