Normal mitochondrial function in the process of metabolic energy production is a key factor in maintaining cellular activities. Many pathological conditions in animals, as well as in patients, are directly or indirectly related to dysfunction of the mitochondria. Monitoring the mitochondrial activity by measuring the autofluorescence of NADH has been the most practical approach since the 1950s. This review presents the principles and technological aspects, as well as typical results, accumulated in our laboratory since the early 1970s. We were able to apply the fiber-optic-based NADH fluorometry to many organs monitored in vivo under various pathophysiological conditions in animals. These studies were the basis for the development of clinical monitoring devices as presented in another article. The encouraging experimental results in animals stimulated us to apply the same technology in patients after technological adaptations as described in the following article (Mayevsky and Barbiro-Michaely 2013b).