To validate the use of ultrafiltration (UF) as an alternative applicable industrial method to replace ultracentrifugation (UC) in the purification of mesenchymal stromal cell (MSC)-secretome. Pharmaceutical formulations containing secretome and/or extracellular vesicles were extracted from adipose-MSCs and bone marrow-MSCs by combining UF or UC with lyophilization. UF led to higher protein, lipid, cytokine and exosomes yields compared with UC. The isolation procedure and cell source influenced immunomodulatory activity, which was in vitro evaluated by inhibition of phytohemagglutinin-activated peripheral blood mononuclear cell proliferation, and by modulation of IL-10, IFN-γ and IL-6. A secretome dosage was identified to obtain the same immunomodulatory activity of MSCs, paving the way for cell-free therapy.