A shotgun proteomics approach that involves multidimensional liquid chromatography coupled to tandem mass spectrometry and database searching techniques was utilized to carry out quantitative proteomic analyses for several Drosophila models of Parkinson’s disease (PD). Utilization of the peptide hits technique for an initial investigation of protein expression alteration across the adult lifespan of a transgenic A30P α-synuclein Drosophila PD model provided evidence for dysregulation of a group of proteins associated with the actin cytoskeleton and the mitochondrion at the presymptomatic and early disease stages that may presage the development of later symptoms. The alterations in the actin cytoskeleton may represent a novel pathogenesis in PD, in addition to the well-accepted mitochondrial dysfunction, oxidative stress, and impairment in the ubiquitin-proteasome system (UPS). This finding led to a comprehensive and sophisticated presymptomatic study of several other closely-related Drosophila PD models including A53T α-synuclein, wild-type (WT) α-synuclein, and parkin null mutants resembling autosomal recessive juvenile parkinsonism (ARJP) utilizing a combination of stable isotope labeling and label-free proteomics approaches to examine causal factors in PD. Proteome measurements of the A53T and WT α-synuclein Drosophila models utilizing the global internal standard technology (GIST) also revealed alterations in the actin cytoskeletal proteins, supporting the association between different forms of α-synuclein and the actin cytoskeleton. Quantitative proteomics of Drosophila parkin null mutants utilizing the GIST strategy and the ion current label-free approach discovered a group of proteins involved in the energy metabolism-associated UPS pathway, suggesting that the impairment in the UPS pathway plays a major role in parkin-linked ARJP. A comparison of the dysregulated proteins in the gain-of-function (A30P, A53T, and WT) α-synuclein Drosophila PD model with that in the loss-of-function parkin null ARJP model shows six commonly perturbed proteins; however, all six proteins exhibit opposite regulation directions. These new findings suggest that α-synuclein and Parkin may be mechanistically linked in certain common molecular pathways underlying PD. Some dysregulated proteins (e.g., ATP synthase β and superoxide dismutase 2) observed at the presymptomatic stage of these Drosophila PD models that were also observed at the postmortem brains from PD patients may provide diagnostic clues to PD.