Mitochondria are complex and dynamic organelles that are essential to the survival of nearly every eukaryotic cell. To generate a foundation for systematic investigations of mitochondrial function and adaptation, we recently established a protein compendium of these organelles across a wide range of tissues. This resource, termed MitoCarta, provides a robust, yet static view of the mitochondrial proteome. We are now applying MitoCarta as a framework for quantifying how mitochondrial proteins and post‐translational modifications (PTMs) change during metabolic perturbations, and to elucidate the roles of these changes in regulating mitochondrial activity. To do so, we blend large‐scale mass spectrometry‐based proteomics with focused biochemistry and molecular biology approaches. In particular, we have recently taken this approach to capture the mitochondrial proteome dynamics during fasting, the onset of obesity, aging, caloric restriction and acute iron deprivation. Our analyses have revealed hundreds of dynamic phosphorylation and acetylation events and have enabled the production of quantitative, searchable maps of mitochondrial alterations across a spectrum of metabolic states. We have leveraged these data to demonstrate that key steps in ketogenesis and branched‐chain amino acid degradation are regulated by reversible PTMs. Moving forward, we plan to further elucidate the mitochondrial signaling network by identifying the regulatory enzymes responsible for managing mitochondrial PTMs, and to define the functions of additional mitochondrial PTMs that are altered in human disease.