Nitric oxide (NO) is a reactive radical synthesized by members of the NO synthase (NOS) family, including mitochondrial-specific NOS (mtNOS). Some of the assays used for the determination of cytoplasmic NOS activity have been utilized to detect mtNOS activity. However, it seems that many of those assays need to be adjusted and optimized to detect NO in the unique environment of mitochondria. Additionally, most mtNOS detection assays are designed and optimized for isolated mitochondria and may exert inherent pitfalls and limitations once used in living cells. This chapter describes several assays used commonly for mtNOS detection in isolated mitochondria and in mitochondria of live cells. Those include colorimetric and spectrophotometric methods, Griess reaction, radioassay, and polarographic and chemiluminescence assays. It also describes fluorescent-based assays for the detection of mitochondrial NO in live cells. Advantages and limitations of each assay are discussed.