Acute Respiratory Distress Syndrome (ARDS) is a devastating clinical disorder with high mortality rates and no specific pharmacological treatment available yet. It is characterized by excessive inflammation in the alveolar compartment resulting in edema of the airspaces due to loss of integrity in the alveolar epithelial-endothelial barrier leading to the development of hypoxemia and often severe respiratory failure. Changes in the permeability of the alveolar epithelial-endothelial barrier contribute to excessive inflammation, the formation of lung edema and impairment of the alveolar fluid clearance. In recent years, Mesenchymal Stromal Cells (MSCs) have attracted attention as a cell therapy for ARDS. MSCs are known to secrete a variety of biologically active factors (growth factors, cytokines, and extracellular vesicles). These paracrine factors have been shown to be major effectors of the anti-inflammatory and regenerative properties observed in multiple in vitro and in vivo studies. This chapter provides a simple protocol on how to investigate the paracrine effect of MSCs on the alveolar epithelial-endothelial barrier functions.