Adipocyte-derived extracellular vesicles (AdEVs), like other types of extracellular vesicles (EVs), are released from their donor parental cells in varying sizes and large quantities. During this process, diverse cargos are often internalized in the lumen or covalently attached on the surface of these EVs, allowing for their horizontal transfer from adipocytes to non-adipocyte cells. Hence, AdEVs can promote the ability of adipocytes to remodel the tumor microenvironment in cancers affecting, among others, breast and colon tissues by acting as mediators of cell-to-cell communication through the release of bioactive molecules. Cancer-associated adipocytes, for instance, can promote tumor growth, metastasis, and therapeutic resistance by secreting AdEVs carrying oncogenic microRNAs, cytokines, and chemokines, which interact with signaling pathways (e.g., JAK/STAT3 and PI3K/AKT) that lead to tumor formation and cancerous behavior. As the importance of EVs in these processes rapidly gains attention, a reliable method of isolating AdEVs also becomes highly essential. Therefore, this chapter describes the use of size-exclusion chromatography given its ability to preserve vesicle integrity and achieve high sample purity.