Misfolded α-synuclein (αSyn) is the hallmark of α-synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). While seed amplification assays (SAA) have demonstrated ultrasensitive detection of misfolded αSyn, they have been primarily used reliably to provide binary (positive/negative) results for diagnostic purposes. We developed an SAA with enhanced specificity for Lewy-fold α-synucleinopathies and introduced a quantifiable measure correlating with clinical severity. Cerebrospinal fluid (CSF) of 170 patients with neurodegenerative diseases and controls was analyzed. Blinded measurements demonstrated 97.8% sensitivity and 100% specificity for Lewy-fold α-synucleinopathies, correctly identifying PD and DLB while excluding MSA. In addition, we validated the strain specificity of the assay by testing brain homogenates from 30 neuropathologically confirmed cases. A novel Lewy-fold pathology (LFP) score based on positive signals in a dilution series provided a quantitative measure of αSyn seeds. The LFP score significantly correlated with motor and cognitive impairment presented by Hoehn and Yahr stage, MDS-UPDRS III, and MoCA. Longitudinal tracking in seven PD cases showed progressive LFP score increases corresponding with clinical deterioration, highlighting the assay's potential for monitoring disease progression at an individual level. Our Lewy-fold-specific SAA enhances ante-mortem diagnosis and differentiates Lewy-fold α-synucleinopathies from MSA. Unlike previous assays, the LFP score offers a quantitative assessment, showing promise as a progression marker and pharmacodynamic biomarker for αSyn-targeting therapies. This represents an important step toward developing an αSyn SAA that could help to track disease progression quantitatively, with potential applications in both clinical diagnostics and therapeutic trials.