Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt-Jacob disease, fatal familial insomnia, and Gerstmann-Straussler-Sheinker disease. Amyloid fibrils prepared from recombinant PrP in vitro share many features of the infectious prions. These fibrils can be used as a synthetic surrogate of PrP(Sc) for development of prion diagnostics, including generation of PrP(Sc)-specific antibody, for screening of antiprion drugs, or for development of antiprion decontamination procedures. Here, we describe the methods of preparation of prion protein fibrils in vitro and biochemical assays for assessing physical properties and the quality of fibrils.