Microdialysis is a technique that collects extracellular fluid through a semipermeable membrane. Various compounds are obtained with this technique in vivo in free-moving animals. Originally, this technique was developed to measure several biogenic amines in rat brain, for example, dopamine from the striatum, acetylcholine from the cerebral cortex, and noradrenaline from the hypothalamus. Recently, the membrane quality and detection limit of many substances have been improved; thus, the microdialysis techniques are widely used to quantify large molecules in the brain. These molecules include neuropeptides and hormones. In this chapter, we describe a principle of the microdialysis technique, how to prepare a microdialysis system, and how to obtain samples from the brain of free-moving animals.