The infectious agents of prion diseases are unorthodox, and they seem to be composed primarily of a misfolded glycoprotein called the prion protein (PrP). Replication of prion infectivity is associated with the conversion of PrP from its normal, cellular form (PrP^C) into a pathogenic form (PrP^Sc), which is characterized biochemically by relative detergent insolubility and protease resistance. Several techniques have been developed in which PrP^C molecules can be converted into the PrP^Sc conformation in vitro (1–8). These biochemical techniques recapitulate several specific aspects of in vivo prion propagation (1–3), and one method, the protein misfolding cyclic amplification technique, also has been shown to amplify infectivity (5). In this chapter, we describe a method for amplifying PrP^Sc molecules from hamster prions in vitro using purified substrates. Specific protocols for substrate preparation, reaction mixture, and product detection are explained. Purified PrP^Sc amplification assays are currently being used to study the biochemical mechanism of prion formation.