The wing imaginal discs in Drosophila larvae are a pair of sac-like structures that later form the wings of the adult fly. During the past decades, wing discs have been used as a simple and accessible model system, for identifying genes and deciphering signaling cascades that play crucial roles in many aspects of development. In this protocol, we describe a simple method for preparing a cell suspension from wing discs (see Graphical abstract). This method can also be applied to the preparation of single-cell suspensions from other types of Drosophila tissues. When combined with genetic labeling, the dissociated cells are suitable for downstream analysis, such as flow cytometry. This method was recently used to isolate different populations of cells from Drosophila imaginal discs ( Yang et al., 2022 ). Graphical abstract: Procedures to prepare a single-cell suspension from Drosophila imaginal discs. Illustration of the main steps to dissect, temporarily store, and dissociate imaginal discs from Drosophila larvae. Refer to the Procedure section for detailed description of each step.