Drosophila melanogasterimaginal discs are a widely used model system to study signal transduction, developmental, and cell biological processes. An important tool to study these processes is immunolabeling. Fluorochrome-conjugated antibody staining allows the researcher to determine the expression pattern and subcellular localization of several proteins in one imaginal disc. In this chapter, I will describe how to dissect, fix, and immunolabel imaginal discs. The protocol is streamlined so that the whole procedure can be performed in 1 d, including image acquisition.