A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was employed for quantitative proteome analysis of a presymptomatic A53T a-synuclein Drosophila model of Parkinson’s disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 (~71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T a-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP-synthase are known to be directly involved in A53T a-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, Mn superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicates that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicates that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P a-synuclein Drosophila model [J. Proteome. Res. 2007, 6, 348 and J. Proteome. Res. 2007, 6, 3729], that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.