Parkinson's disease and other synucleinopathies are neurodegenerative disorders defined by the pathological aggregation of alpha-synuclein (α-syn). The alpha-synuclein Seed Amplification Assay (α-syn SAA), also known as real-time quaking-induced conversion (RT-QuIC), has gained recognition as a high-sensitivity, high-specificity diagnostic tool capable of detecting misfolded α-syn seeds in biological samples through cyclic amplification and thioflavin T-based fluorescence readout. Despite its diagnostic promise, the lack of standardized protocols across laboratories remains a critical obstacle to clinical implementation. This systematic review, conducted following PRISMA guidelines, analysed 78 studies published between 2019 and 2025 to comprehensively characterize the methodological parameters employed in α-syn SAA. Data extraction encompassed equipment settings (fluorescence reader model, incubation temperature, shaking speed and ratio, bead configuration), reaction mixture components (recombinant α-syn concentration and type, buffer composition, NaCl and SDS concentrations, thioflavin T concentration), sample types, investigated pathologies, and positivity criteria. Findings revealed relative consensus in certain parameters. First, 73% of studies used the same equipment, 0.1 mg/mL recombinant α-syn, and 10 μmol/L thioflavin T; while substantial heterogeneity persisted in incubation temperature, buffer composition, and diagnostic thresholds. Cerebrospinal fluid was the predominant sample type, and Parkinson's disease the most frequently studied condition. Positivity criteria varied widely, with no universally validated standard. These results underscore the urgent need for harmonized protocols to ensure reproducibility and facilitate the clinical translation of α-syn SAA as a reliable biomarker tool to detect synucleinopathies.