The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) is gaining momentum, yet scalable purification remains a key bottleneck. In collaboration with Cytiva, RoosterBio developed a downstream process to enhance EV yield and purity. Conditioned media from hBM-MSCs cultured in 3L bioreactors was treated with Agent V (RoosterBio, patent pending), clarified using optimized filters, and concentrated via tangential flow filtration (TFF). Chromatography using Capto Core 400 and superSEC resins achieved >85% EV recovery and significantly improved purity (>1E11 particles/mg protein). EVs retained identity markers (CD63, CD9, CD81) and CD73 activity, confirming bioactivity. This study presents a robust, scalable EV purification platform suitable for clinical translation.