Due to their high proliferative potential, multipotency, paracrine effect, and immunomodulatory activity, mesenchymal stromal cells (MSC) are ideal candidates for regenerative medicine and immunotherapy. They represent the major stem cells for cell therapy. Currently, MSC are involved in clinical trials as a therapy for immune-related diseases (such as graft versus host disease), bone and cartilage diseases, cardiovascular diseases and neurological diseases. Although most of these studies are still phase I or II trials (according to ClinicalTrials.gov), promising results are already emerging. Due to the prolonged expansion regimens that are needed in the clinic to obtain sufficient amounts of MSCs for therapy, and based on the patient-specific quality of cells, it is quite likely that long-term culture evokes continuous changes in MSCs. In particular, a substantial proportion of cells may undergo senescence. Cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies’ outcomes. In fact, when cells become senescent in vitro they significantly change their phenotype. These changes also affect MSC deeply at a functional level. For instance, late passage MSC were clinically shown to be less effective in ameliorating graft-versus-host disease than early passage cells. It would, thus, be of great significance to monitor the occurrence of a senescent phenotype in MSCs addressed to clinical uses and to evaluate the functional consequences of senescence in MSCs which could affect their clinical therapeutic potential, taking into account their paracrine effects, immunomodulatory activity, differentiation potential, and cell migration ability. Therefore, a need exists to develop reliable and efficient tools to monitor the progression of senescence in MSC, especially in the context of tissue regeneration, for the proper assessment of efficacy, patient inclusion criteria and regulatory/insurance issues Understanding the mechanisms that drive toward MSC growth arrest and evaluating how to counteract these for preserving a functional MSC pool is of fundamental importance for the development of efficient cell-based therapeutic approaches. An updated critical presentation of the possible strategies for in vitro senescence monitoring and prevention constitutes the aim of this presentation.