Summary Many cases of Parkinson’s disease (PD), and even more so atypical parkinsonian disorders, are misdiagnosed. Misdiagnosis not only causes high stress and anxiety to patients, families, and caregivers, but also is a major impediment to developing effective therapy for these diseases. Recently, we have demonstrated that the α- synuclein concentration in extracellular vesicles (EVs) immunoprecipitated from serum or plasma using oligodendroglial and neuronal markers, and in particular the ratio between the α-synuclein concentrations in the two types of EVs, provided a sensitive biomarker for distinguishing between Parkinson’s disease and multiple system atrophy (MSA). This liquid biopsy approach requires only a minimally invasive blood draw and could lead to a major advancement in developing diagnostic tests for these diseases. However, the definition of the groups was based on clinical diagnosis, which is error-prone, creating a chicken-and-egg problem. To address this issue, here we propose a pilot study testing biomarkers in serum samples collected postmortem for which the diagnosis was validated pathologically. This strategy could not work for total α-synuclein for specific reasons that are hypothesized not to be applicable to the biomarkers in the current proposal. We will also test the utility of each of the new biomarkers as part of diagnostic biomarker panel for distinguishing PD, MSA, and two additional atypical parkinsonian syndromes—progressive supranuclear palsy and corticobasal syndrome—from each other and from control samples. The main goal of the pilot study is to determine whether pathologically validated postmortem samples can be used for biomarker measurement in CNS-originating EVs for parkinsonian syndromes. A secondary goal is to test the contribution of each biomarker to the diagnostic panel. The study has the potential to lead to future development of minimally invasive biomarkers allowing early and accurate diagnosis of parkinsonian disorders.