Mitochondria play a critical role in many different pathologic conditions. Increasing evidence has shown that mitochondrial reactive oxygen species (ROS) production may provide an etiologic link between mitochondria and pathologics. The widespread use of laboratory mice as models for a host of human diseases makes the quantification and localization of ROS production from mice an important endeavor. This chapter presents approaches to the quantification and localization of ROS from mouse brain, liver, and beta cell mitochondria. Techniques for the isolation of mitochondria and mitochondrial fractions and the subsequent quantification of ROS with Amplex Red or a FACS-based method on intact cells are described.