Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder caused by dopaminergic neuronal deaths. For a proper treatment of PD, biomarkers that would enable us to monitor disease progression or responses to a disease-modifying agent, or that can be used as a presymptomatic marker are critical. In addition, a biomarker that could be used to distinguish between the various diseases that manifest with PD-like symptoms would greatly enhance patient care and management of future clinical trials. Previous research indicates that c-Abl is overactive in PD patients resulting in an increased phosphorylation of the tyrosine 39 residue of alpha-synuclein (a-syn) (pY39 a-syn) and eventually dopaminergic neuronal death via a-syn aggregation. For this reason, monitoring pY39 a-syn will enable us to diagnose presymptomatic PD, monitoring disease progression and monitoring the response disease-modifying agents. We hypothesized that this increased phosphorylation on Y39 of a-syn (pY39 a-syn) will be reflected in the cerebrospinal fluid (CSF) of PD patients and monitoring pY39 a-syn level in CSF will enable us to monitor pY39 a-syn level in the brain. Mass spectrometry has been widely used for the detection of phosphorylated peptides using parallel reaction monitoring (PRM) and we utilized the PRM mass spectrometry for the detection of pY39 a-syn. However, pY39 a-syn was not detected even after immunoprecipitation of a-syn, phosphopeptide enrichment or phosphotyrosine peptide enrichment from a practical volume. For this reason, we have developed a two-step enrichment method in which pY39 a-syn was first enriched with an anti-phosphotyrosine antibody followed by a further enrichment of phosphopeptides with titanium oxide (TiO2) beads to remove non-phosphopeptides. Accurate quantification was obtained by including a synthetic pY39 a-syn peptide labeled with heavy lysine that was added before an enzyme digestion. To detect pY39 α-syn peptide in CSF lower than 10 femtomolar concentration, experimental parameters were optimized. Comparison of pY39 a-syn to total a-syn in CSF from PD patients and controls reveals that pY39 a-syn levels are significantly elevated in the CSF of PD patient versus controls. This optimized two-step enrichment PRM detection method may be of value as a biomarker to monitor c-Abl activation in PD patients for future diagnostic studies as well as a pharmacodynamic marker for c-Abl therapeutic trials in PD. Furthermore, this developed method can be applicable to quantifying other phosphotyrosine peptides with low abundance in biological samples.