Parkinson's disease (PD), dementia with Lewy bodies (DLB), and other synucleinopathies are characterized by the accumulation of abnormal, self-propagating aggregates of α-synuclein. RT-QuIC or seed amplification assays are currently showing unprecedented diagnostic sensitivities and specificities for synucleinopathies even in prodromal phases years in advance of the onset of Parkinsonian signs or dementia. However, commonly used α-synuclein seed amplification assays take ≥48 h to perform as applied to patients' diagnostic biospecimens. Here, we report the development of a faster α-synuclein RT-QuIC assay that is as analytically sensitive as prior assays of this type, but can be completed in ≤12 h for brain, skin, and intestinal mucosa, with positive signals often arising in <
5 h. CSF assays took a few hours longer. Our same-day α-synuclein RT-QuIC (sdRT-QuIC) assay should increase the practicality, cost-effectiveness, and throughput of measurements of pathological forms of α-synuclein for fundamental research, clinical diagnosis, and therapeutics development.