Reactive oxygen species (ROS) are constantly produced by skeletal muscle and this production is increased during contractile activity. Understanding the role that ROS play in skeletal muscle requires an understanding of the species of ROS produced, the subcellular site of production, the time-course of ROS production, and the effects of inhibiting these ROS using specific antioxidants or inhibitors. Unfortunately, due to the extremely short half-lives of ROS, many methods to study ROS have had to rely on downstream markers of ROS reactions which cannot provide specific information. In vivo microdialysis is one technique that allows access to a specific site of ROS production allowing the continuous measurement of ROS at rest and during contractile activity. This chapter will describe the technique of microdialysis to measure ROS in skeletal muscle as well as discussing specific methods to detect superoxide, nitric oxide, and hydroxyl radical activity using in vivo microdialysis in skeletal muscle at rest and during contractile activity.