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Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are processed by Furin. To quantitatively assess the potential role of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a simple assay by combining genetics, ex vivo organ culture, pharmacological treatment, and imaging analyses. The protocol herein describes how to ex vivo culture Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over a long period of time in the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl parts using microscopy. Although the assay described here is for assessing the effect of Furin inhibition on Bnl cleavage in the Drosophila larval wing imaginal disc, the principle and methodology can easily be adopted for any other signals, tissue systems, or organisms. This strategy and protocol provide an assay for examining Furin activity on a specific substrate by directly visualizing the spatiotemporal distribution of its truncated parts in an ex vivo-cultured organ.