Cardiosphere-derived cells (CDCs) and their extracellular vesicles (CDC-EVs) can rejuvenate and improve cardiac function, but their efficacy varies among donors. This study aimed to identify predictors of CDC- EV potency. CDCs from human donors were analyzed for phenotypical and biological properties. The anti- aging activity of CDC-EVs was tested using an in vitro matrix assay. Results showed that donor age did not determine potency, but CDC senescence correlated with most bioactive properties. However, senes- cence alone was insufficient to predict CDC-EV potency. CDC-EVs were classified as more potent (P-EVs) or less potent (NP-EVs) based on assay performance. In a rat model of premature cardiac aging, P-EVs reduced senescence-associated GLB1 gene expression and protected against hypertrophy and fibrosis, while NP-EVs had negative effects. The study concludes that CDC-EV anti-aging potency cannot be pre- dicted by donor age or CDC senescence, proposing an in vitro potency assay for evaluating CDC-EVs before clinical use.