Updated project metadata. Mitochondria perform central functions in cellular bioenergetics, metabolism and signaling and their malfunction has been linked to numerous diseases. The available studies cover only part of the mitochondrial proteome and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative, quantitative MS-based experimental approach to define the high confidence proteome of yeast mitochondria and to identify new mitochondrial proteins. The analysis includes protein abundance under fermentable and non-fermentable growth, submitochondrial localization, single-protein analysis and subcellular classification of mitochondria-associated fractions. We identified novel mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS) and coenzyme Q biosynthesis cluster as well as new mitochondrial proteins with dual cellular localization. The integrative proteome provides a high confidence source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment.