Abstract: Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of ?-synuclein (?-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ~4% of ?-syn is phosphorylated at Ser129 (pS129-?-syn), whereas >90% pS129-?-syn may be found in LBs. Embodiments of the invention include a duplex assay for both total ?-synuclein and pS129-?-synuclein, which allows measuring both analytes in the same sample, leading to substantial saving in sample volume. The assays can be widely used in methods for detecting pS129-?-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.