Sirtuins (SIRT1-SIRT7) are a family of NAD(+)-dependent protein deacetylases that regulate cell survival, metabolism, and longevity. SIRT3 is localized to the mitochondria where it deacetylates several key metabolic enzymes: acetylcoenzyme A synthetase, glutamate dehydrogenase, and subunits of complex I and thereby regulates their enzymatic activity. SIRT3 is therefore emerging as a metabolic sensor that responds to change in the energy status of the cell via NAD(+) and that modulates the activity of key metabolic enzymes via protein deacetylation. Here we review experimental approaches that can be used in vitro and in vivo to study the role of acetylation in mitochondrial cell biology.