•Evaluation and optimization of different nuclease alternatives.•MNase – the top performer nuclease– removes more than 60% of DNA.•Anion exchange chromatography is effective for extracellular vesicles purification.•Capto™ Q ImpRes – the best resin – has a recovery yield higher than 85%.•Isolation of purified MSC-derived extracellular vesicles, with contaminant levels below regulatory agencies's limits. Extracellular vesicles (EVs) have emerged as a novel cell-free strategy with broad biomedical applications in disease diagnosis and prognosis as well as therapy. Moving EVs to the clinics will require the development of cost-effective manufacturing processes capable of delivering a high-quality product with high yield. In this work, we explore the purification of EVs from conditioned media derived from human mesenchymal stromal cell bioreactor cultures using a nuclease digestion followed by anion exchange (AEX) chromatography as a scalable purification platform. The performance of different nucleases was evaluated and optimized, with MNase being the top performer. Concerning the chromatographic step, all anion-exchange resins successfully isolated EVs, being Capto™ Q ImpRes the lead resin with recovery yields exceeding 85% concomitant with removal of more than 98% of proteins and host cell DNA. Overall, our results show that the purification platform proposed in this work is a viable alternative to the existing methods, capable of delivering highly purified EVs and able to meet the requirements imposed by the regulatory agencies for clinical trial testing, and that can contribute for the integration of EVs-based therapies in the clinic.