Using this novel approach, we have generated a streamlined RT-QuIC assay that (1) is able to measure the α-synuclein seeding of 96 MSA samples, run in quadruplicate, in less than 48 h; (2) requires the use of a minimal amount of commercially available recombinant α-synuclein monomer (5 μg) per well; (3) requires the use of a minimal amount of brain material (5 μg); (4) generates RT-QuIC-derived α-synuclein fibrils that are conformationally distinct between patients with different synucleinopathies; and (5) is capable of subtyping MSA brains according to their α-synuclein seeding behavior. Here, SNCA is linked to multiple system atrophy.