Recent biochemical and genetic approaches have clearly defined the functional role of critical components in tyrosine phosphorylation-dependent signal transduction. These signaling modulators often exhibit evolutionarily conserved functions across various species. It has been proposed that if protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and thousands of their substrates could be identified and characterized, it would significantly advance our understanding of the underlying mechanisms that control animal development and physiological homeostasis. The fruit fly Drosophila melanogester has been used extensively as a model organism for investigating the developmental processes, but the state of its tyrosine phosphorylation is poorly characterized. In the current study, we used advanced mass spectrometry (MS)-based shotgun analyses to profile the tyrosine phosphoproteome of Drosophila S2 cells. Using immunoaffinity isolation of the phosphotyrosine (pTyr) subproteome from cells treated with pervanadate followed by enrichment of phosphopeptides, we identified 562 nonredundant pTyr sites in 245 proteins. Both this predefined pTyr proteome subset and the total cell lysates were then used as sample sources to identify potential substrates of dPTP61F, the smallest member in terms of amino acid number and molecular weight in the Drosophila PTP family and the ortholog of human PTP1B and T Cell-PTP, by substrate trapping. In total, 20 unique proteins were found to be specifically associated with the trapping mutant form of dPTP61F, eluted by vanadate (VO4(3-)), and identified by MS analyses. Among them, 16 potential substrates were confirmed as tyrosine phosphorylated proteins, including a receptor PTK PDGF/VEGF receptor, a cytosolic PTK Abl, and several components of SCAR/WAVE complex, which may work in coordination to control actin dynamics. Thus, our data suggest that dPTP61F plays a central role in counteracting PTK-mediated signaling pathways as well as in regulating actin reorganization and remodeling through tyrosine dephosphorylation of critical substrates in Drosophila cells.