Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. The present review aims: 1. To provide an historical overview of NADH fluorescence monitoring and its physiological significance. 2. To present the solid scientific ground underlying NADH fluorescence measurements based on published materials. 3. To provide the reader with basic information on the methodologies used in the past and the current state of the art fluorometers. 4. To clarify the various factors affecting monitored signals, including artifacts. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To better understand the tissue function, the multiparametric approach has been developed where NADH serves as the key parameter. The development of new light sources in UV and visible spectra, has led to the development of small compact units applicable in clinical conditions for better diagnosis of patients.