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Summary: Exosomes are a class of extracellular vesicles that play a role in intercellular signaling under diverse contexts. Here, we describe a protocol that has been optimized for the isolation and characterization of exosomes from a Drosophila melanogaster cell line using size exclusion chromatography (SEC). The specific focus of this protocol was to examine the starvation-induced exosome loading of a protein.For complete details on the use and execution of this protocol, please refer to Pandey et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.