Establishing reproducible aggregation kinetics and amyloid formation of α-synuclein (α-Syn) is of great interest for understanding Parkinson’s disease (PD) pathogenesis. α-Syn, 140 amino acid residues intrinsically disorder protein (IDP), is well known for its inconsistent aggregation behaviour in vitro. Previously different methods/conditions like orbital agitation, usage of small glass beads in plate reader assay were reported to achieve higher reproducibility. In addition to mechanical agitation, here we report the usage of aggregate free low molecular weight (LMW) solution as a starting material for monitoring aggregation pathway of α-Syn. This allowed us to obtain the reproducible fibrillation kinetics of α-Syn at a satisfactory level. This LMW could be used not only for understanding PD pathogenesis but also for screening inhibitors against α-Syn fibrillation and to study many molecular events in vitro.