The involvement of mitochondrial dysfunction in many pathophysiological conditions and human diseases is well documented. In order to evaluate mitochondrial function in vitro,many experimental systems have been developed. Nevertheless the number of in vivo monitoring systems for the evaluation of mitochondrial activities in intact animals and patients is relatively limited. The pioneering development of the conceptual and technological aspects ofmitochondrial monitoring, in vitro and in vivo, was done by the late Prof. Britton Chance (July 24, 1913-November 16, 2010) since the early 1950s. It was my privilege to join his laboratory in 1972 and collaborate with him for almost four decades. The main achievements of our collaboration are presented in this paper. Our activities included cycles of technology development, followed by its applications to study various pathophysiological conditions. In the initial stage, the first fiber-optic-based NADH fluorometer was developed. This device enabled us to monitor various organs in anesthetized animals aswell as the brain of nonanesthetized small animals. Later on, the addition of various physiological parameters to NADH monitoring enabled us to correlate mitochondrial function with other cellular functions. The application of the developed technology to clinical situations was a major interest of Prof. Chance and indeed this goal was achieved in the last decade.As of today, the basic tool forNADHmonitoring and the large database of results are available for large-scale experimental and clinical applications.