ABSTRACT: Prion diseases are caused by the conversion of normal cellular prion proteins (PrP) into lethal prion aggregates. These prion aggregates are composed of proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-prion pharmaceuticals available to treat or prevent prion disease. Methods of discovering anti-prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-prion molecules: Congo red, curcumin, GN8, quinacrine, chloropromazine, tetracycline, and TUDCA (taurourspdeoxycholic acid), as well as four suspected anti-prion compounds: trans-resveratrol, rosmarinic acid, myricetin and ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-prion fibril compounds. Abbreviations: PrP, prion protein; PK, proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy; TUDCA, taurourspdeoxycholic acid; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; CJD, Creutzfeldt Jakob disease; GSS, Gerstmann-Sträussler-Scheinker syndrome; FFI, fatal familial insomnia; PrPc, cellular prion protein; recPrPC, recombinant monomeric prion protein; PrPSc, infectious particle of misfolded prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA, Protein Misfolding Cyclic Amplification; LPS, lipopolysaccharide; EGCG, epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide; DMSO, dimethyl sulfoxide; ScN2A, scrapie infected neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP 23-231, recombinant full-length mouse prion protein residues 23-231; EDTA; PICUP, photo-induced cross-linking of unmodified protein; BSA, bovine serum albumin;; PMSF, phenylmethanesulfonyl fluoride.