Ex vivo calcium imaging in Drosophila opens an expansive amount of research avenues for the study of live signal propagation through complex tissue. Here, we describe how to isolate Drosophila organs of interest, like the developing wing imaginal disc and larval brain, culture them for extended periods, up to 10 h, and how to image the calcium dynamics occurring within them using genetically encoded biosensors like GCaMP. This protocol enables the study of complex calcium signaling dynamics, which is conserved throughout biology in such processes as cell differentiation and proliferation, immune reactions, wound healing, and cell-to-cell and organ-to-organ communication, among others. These methods also allow pharmacological compounds to be tested to observe effects on calcium dynamics with the applications of target identification and therapeutic development.