Human reticulocytes were enriched from human umbilical blood, and cultured in vitro for the production of reticulocyte-derived exosomes (HuRex)to unveil their proteomic composition. As research on the molecular-cargo of exosomes is largely confounded by the lack of a “gold-standard” methodology, we applied several approaches for obtaining HuRex prior to MS. HuRex preparations were obtained from cultures in absence of serum (AS) from three donors and isolated by means of size-exclusion chromatography (SEC) (n=3) and ultracentrifugation (UC) (n=3). HuRex preparations in the presence of serum were obtained from three other donors and purified by SEC (n=3) and UC (n=1).