Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.