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Summary: Many genetically encoded tools, including large collections of GAL4 transgenic lines, can be used to visualize neurons of the Drosophila melanogaster brain. However, identifying transgenic lines that are expressed sparsely enough to label individual neurons, or groups of neurons that innervate a particular brain region, remains technically challenging. Here, we provide a detailed procedure in which we used broadly expressed transgenic lines and two-photon microscopy to photo-label neurons with specificity, thereby permitting their morphological characterization.For complete details on the use and execution of this protocol, please refer to Li et al. (2020).