α-synuclein (aSyn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of aSyn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies towards aSyn PTMs. Here, by taking advantage of aSyn thermostability, we applied a method to achieve high enrichment of soluble aSyn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% aSyn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal-acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of aSyn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies. © 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12251.