ABSTRACTα-Synuclein is an intrinsically disordered protein that forms amyloids in Parkinson’s disease. Currently, detection methods predominantly report on the formation of mature amyloids but have poor sensitivity to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitorα-synuclein oligomer formation using the fluorescence lifetime of molecular rotors. We detect oligomer formation and conversion into amyloids forwild typeand twoα-synuclein variants; the pathological mutant A30P and ΔPα-synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30Pα-synuclein shows a similar rate of oligomer formation compared towild type α-synuclein, whereas ΔPα-synuclein shows delayed oligomer formation. Additionally, both variants demonstrate a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism ofα-synuclein aggregation which is key to understanding the pathology of Parkinson’s disease.