Research on mesenchymal stromal cells (MSC) has progressed significantly over recent decades, transitioning from a focus on their differentiation potential to the mechanisms underlying their paracrine action involving growth factors, cytokines, chemokines, and extracellular vesicles (EV) present in MSC-conditioned medium (CM), all of which play key roles in MSC therapeutic properties. This paradigm shift has paved the way for a new cell-free therapeutic approach, beginning with MSC-CM and later evolving to MSC-EV isolated from CM, which have demonstrated therapeutic properties similar to their parent cells in certain applications. MSC and MSC-EV have been explored for a wide range of therapeutic applications, with promising safety profiles and potential efficacy. However, significant challenges remain, particularly the need for optimization in key areas to advance MSC and MSC-derived products as cell-based therapies. A critical hurdle is the development of robust and standardized potency assays to quantitatively and functionally evaluate their regenerative properties. Several assays have been proposed to assess MSC and MSC-CM capabilities, including their promotion of migration, angiogenesis, immunomodulation, and control of oxidative stress. Findings suggest that employing a matrix of multiple well-defined potency assays could address the variability introduced by differences in protocols and donors, while also helping to elucidate the relevant mechanisms of action for both MSC and MSC-EV in various therapeutic contexts.