Mesenchymal Stem Cells (MSCs) are widely recognized for their immunomodulatory and regenerative properties. In recent years, extracellular vesicles (EVs) derived from MSCs have garnered significant attention for replicating many of the regenerative functions of their parent cells. EVs can be classified based on their origin and biogenesis into exosomes (endosomally derived), ectosomes (budding from the plasma membrane), and apoptotic bodies. They can also be categorized by size into small EVs (<200 nm), large EVs (>200 nm), and apoptotic bodies (≤1 μm). Among these, small EVs, particularly exosomes, are most prominently associated with regenerative and immunomodulatory roles. Despite their potential, the translation of MSC-derived EVs into clinical applications remains limited. A major challenge lies in the lack of standardized protocols for isolating and characterizing EVs from MSC-conditioned media. This chapter explores the hurdles in standardizing these techniques and proposes key factors to optimize the isolation and characterization processes, bridging the gap between their therapeutic potential and clinical implementation.