Homeostasis of human and animal organisms and its disturbance are explored mainly by evaluating metabolic substrates and metabolite concentrations and their changes under different clinical conditions. This review describes a method of tissue biochemistry monitoring by analysis of extracellular fluid dialysate. Microdialysis not only provides information about the general disturbances of the body, but allows insight into the local metabolism of tissues and organs. It can be applied both in experimental conditions (e.g. animal neurophysiology) and in clinical practice (episodes of brain ischemia, glycemic disturbances, pharmacodynamic studies). The technique of microdialysis is relatively simple and is based on microdialysis probe implantation into the studied organ or tissue, followed by measurement of the obtained samples of extracellular fluid dialysate. During the last twenty years, microdialysis probes have been inserted into different part of the living organism: brain, skeletal muscles, subcutaneous adipose tissue, transplanted tissues (myocutaneous flap) and organs (liver), tendons, skin, breast gland, lungs, heart muscle, intestines, and body cavities. The possibility of frequent sampling (every 20-60 minutes) and measuring the concentrations of glucose, glycerol, lactates, and pyruvates with a bedside analyzer in an average time of 2-3 minutes is considered a great advantage of this method. Microdialysis gives many opportunities for a better understanding of how living organisms are functioning, but it requires cautious application and validation in clinical observations in order to make standards and recommendations (diseases and clinical conditions, substances for monitoring, referenced values and trends) for its common medical usage.