AbstractDespite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size‐exclusion chromatography (DG‐SEC), differential ultracentrifugation (dUC) and/or stand‐alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer‐associated fibroblasts (CAF). EV‐enriched but protein‐depleted versus EV‐depleted but protein‐enriched DG‐SEC fractions, and EV‐containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose‐dependently evaluated for monocyte‐derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG‐SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF‐A and MCP‐1 and potently stimulated MoDC maturation. In contrast, EV‐containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose‐dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method‐dependent off‐target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration.