Synucleinopathies are a group of neurodegenerative disorders characterized by the accumulation of α-synuclein-rich Lewy pathology within neurons and/or glia. Up to 90% of α-synuclein protein within Lewy pathology is phosphorylated at serine residue 129 (pS129), prompting the development of technologies for measuring α-synuclein phosphorylation as a proxy for pathological burden in these disorders. While several assays can quantify human pS129 α-synuclein in patient brain tissues and biofluids, none cross-react with mouse α-synuclein, which is problematic considering the preponderance of mouse models at the forefront of α-synuclein research. In this project, we utilized a bead-based assay technology, AlphaLISA® SureFire® UltraTM, to address this unmet need. Our work included reformulating two existing total and pS129 α-synuclein assay kits to yield robust and ultrasensitive (LLoQ ≤0.5pg/mL) quantification of mouse and human wild-type and pS129 α-synuclein protein, which we then employed to assess α-synuclein S129 phosphorylation in different mouse brain tissue preparations. We found α-synuclein S129 phosphorylation was remarkably consistent across 10 regions of the C57BL/6 mouse brain (~0.15% total α-synuclein), with >99.5% pS129 α-synuclein localized to the cytosol. This profile changed considerably upon bilateral intrastriatal inoculation of these mice with wild-type mouse α-synuclein pre-formed fibrils, which increased the proportion of α-synuclein phosphorylation up to 6.1-fold in some brain regions, primarily due to greater proportions of membrane-bound (1-7%) and detergent-insoluble (18-30%) pS129 α-synuclein. These changes were accompanied by significant cytosolic and membrane-bound aggregated α-synuclein. Overall, we highlight the potential for these new immunoassays to advance knowledge surrounding α-synuclein phosphorylation and synucleinopathy pathogenesis.