Our study introduces a novel methodology for generating bona fide infectious prions de novo, enabling a comprehensive analysis of protein misfolding across the largest collection of prion proteins from mammalian species to date. Using the Protein Misfolding Shaking Amplification (PMSA) technique, we tested hundreds of different recombinant prion proteins, classifying them according to their spontaneous misfolding propensity and conformational variability. This method has successfully yielded infectious prions from an extraordinary range of species, including bat, deer, sheep, cow, mink, pig, human, dog, rabbit, and rodents, among hundreds of others. We ranked the misfolding propensities of all known wild-type prion proteins and analyzed the theoretical stability of their globular isoforms to explore potential correlations with misfolding propensity. The authenticity of the infectious prions generated through our methodology was validated through comprehensive inoculation experiments that confirmed their infectivity. The notable efficiency of our method in replicating spontaneous prion misfolding provides a valuable opportunity to address fundamental questions in the prion research field, such as defining infectivity determinants, interspecies transmission barriers, and the structural influence of specific amino acids. This comprehensive framework will establish the foundation for significant progress in prion research and provide invaluable information for future diagnostic and therapeutic applications.