Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals.