Prion diseases are caused by proteinaceous pathogens termed prions. Although the details of the mechanism of prion propagation are not fully understood, conformational conversion of cellular prion protein (PrP^C) to misfolded, disease-associated scrapie prion protein (PrP^Sc) is considered the essential biochemical event for prion replication. Currently, studying prion replication in vitro is difficult due to the lack of a system which fully recapitulates the in vivo phenomenon. Over the last 15 years, a number of in vitro systems supporting PrP^C conversion, PrP^Sc amplification, or amyloid fibril formation have been established. In this review, we describe the evolving methodology of in vitro prion propagation assays and discuss their ability in reflecting prion propagation in vivo.