Mesenchymal stem cells (MSCs) show therapeutic potential for inflammatory diseases; however, their efficacy is often compromised during ex vivo expansion. Here, melatonin (MT) is identified as a safe and effective priming agent enhancing MSC functionality through intracellular glutathione dynamics. Using MitoFreSHtracer, a real-time quantitative monitoring platform is developed for live MSCs under ferroptotic stress. Multiple parameters based on the fluorescence ratio mean, heterogeneity, and ferroptotic stress resistance capacity are used. Dynamic assessment of MSC heterogeneity related to ex vivo expansion and accurate key functional capacity predictions are established in MSC batches and tissue sources. High-throughput screening of 39 flavonoids and 7 antioxidants identifies MT as a pharmacopeia-compliant priming agent with superior redox-enhancing capacity. MT priming robustly activates CREB1–NRF2 and JAK–STAT3–PD-L1 pathways, leading to the enhanced clonogenicity, migration, angiogenesis, anti-inflammation, and immunosuppressive properties. Transcriptomic analysis revealed molecular signatures associated with immune modulation and tissue regeneration. MT-primed MSCs significantly alleviates Th2-mediated inflammation and improves lung engraftment in allergic asthma mice. A deep learning-based convolutional neural network for automated bronchoalveolar lavage fluid analysis provides objective and efficient inflammatory cell profiling. Overall, an integrated approach involving MT advances the development of high-quality MSC-based therapies for asthma and other inflammatory diseases.