Mitochondrial membrane potential (Delta Psi(m)) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to Delta Psi(m), and partially to plasma membrane potential (Delta Psi(p)), for non-invasive, longitudinal monitoring of Delta Psi(m) in vitro and in vivo. We applied this new technology to evaluate the aging-related change of Delta Psi(m) in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of Delta Psi(m) in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.