Prion diseases (PrDs) are devastating and fatal conditions characterized by the accumulation of the misfolded prion protein (PrPSc) in the central nervous system (CNS). Definitive diagnosis of PrDs relies on the detection of prions in CNS tissues collected postmortem. The advent of a highly sensitive cell-free amplification technique, named protein misfolding cyclic amplification (PMCA), has revolutionized this field. It has revealed trace amounts of prions in various tissues, including cerebrospinal fluid, urine, blood, and olfactory mucosa of patients with different forms of PrDs. PMCA mirrors in vitro the pathological process of protein misfolding and aggregation, which occurs in vivo but in a significantly accelerated manner. For this reason, this technology is currently used in specialized laboratories to support research and diagnostic activities in human and animal PrDs. This chapter highlights the latest advances and applications of PMCA in the diagnosis of human PrDs.