Phosphorylated α-synuclein (PS-129), a protein implicated in the pathogenesis of Parkinson’s disease (PD), was identified by mass spectrometry in human cerebrospinal fluid (CSF). A highly sensitive and specific assay was established and used to measure PS-129 together with total α-synuclein in the CSF of patients with PD, other parkinsonian disorders such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), and healthy individuals (a total of ~600 samples). PS-129 CSF concentrations correlated weakly with PD severity and, when combined with total α-synuclein concentrations in CSF, contributed to distinguishing PD from MSA and PSP. Further rigorous validation in independent cohorts of patients, especially those where samples have been collected longitudinally, will determine whether the concentration of PS-129 in CSF will be useful for diagnosing PD and for monitoring PD severity and progression.