ABSTRACT Parkinson’s disease (PD) is a neurodegenerative disorder that is typically diagnosed when 60% of a brain region’s dopaminergic neurons have already degenerated. A strong contender for the elusive molecular biomarker is a neuronal protein - α-synuclein and its post-translationally modified phosphorylated isoform (pSer-129). Along with other C-terminal modifications, this modification has been found as the insoluble aggregated and fibrillar form in the brain tissue of deceased Parkinson’s patients. The progression and disease severity has been correlated to its relative levels in patient’s cerebrospinal fluids (CSF) as well. However, current technologies, such as immuno-assays and mass-spectrometry lack the necessary sensitivity and capability to accurately quantify both the phosphorylated and unmodified form of the protein simultaneously and in clinically limited samples, such as the cerebrospinal fluid. This technological limitation has hampered the development of a robust clinical assay and validation of these implicated neuronal biomarkers. Fluorosequencing, a new single molecule protein sequencing technology, is a massively parallelized platform for identifying and quantifying individual proteins in a complex mixture. The important and distinguishing feature of the single molecule platform is its inherent ability to absolutely quantify and discriminate the different species of peptides, including the species differing in the positions and the occupancy of phosphorylated residues. Through the grant phase I and II, we propose to utilize this platform technology to develop a clinical assay for measuring the abundances of the different species of α-synuclein from extremely limited biological samples and correlating the measurements with patient outcomes. In phase I, we will spike-in modified and unmodified alpha-synuclein species at different proportions into CSF, establishing assay reproducibility and replicability (aim 1) and obtain limits of detection and discrimination between the different α-synuclein phosphorylated species (aim 2). Completing these objectives will establish an optimized work-flow and the determination of the sample requirements necessary for measuring the levels in patient samples. Successful completion of the two phases would result in a commercialization path towards a laboratory developed test or diagnostic assay.