This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. Methods to assess mitochondrial function is of great interest to neuroscientists studying chronic forms of neurodegeneration, including Parkinson’s, Alzheimer’s, ALS, Huntington’s and other triplet repeat diseases, but also to those working on acute conditions such as stroke and traumatic brain injury. This volume covers research methods on how to assess the life cycle of mitochondria including trafficking, fusion, fission, and degradation. Multiple perspectives on the complex and difficult problem of measurement of mitochondrial reactive oxygen species production with fluorescent indicators and techniques ranging in scope from measurements on isolated mitochondria to non-invasive imaging of metabolic function.