Protein Misfolding Cyclic Amplification (PMCA) has proved to be an efficient method mimicking in vitro some of the fundamental steps involved in prion replication in vivo. Thus, it can be used to efficiently replicate a variety of prion strains/species. The in vitro generated prions possess key prion features, i.e., they are infectious in vivo and maintain their strain specificity. One of the big challenges is its use for studying prion transmission barriers. PMCA has been efficiently used for adapting different prion species through a range of species barriers; however its capacity for overcoming purportedly unbreakable species barriers compels us to adapt it in order to use it as a reliable technique. In addition, this in vitro method might be a crucial tool in evaluating the potential risks of different prion strains (natural or experimentally generated in vitro) to humans and animals.