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.