Mitochondria perform several fundamental cellular processes in higher eukaryotes including oxidative phosphorylation, Fe/S cluster formation and apoptosis. Dysfunction of the organelle is associated with a wide range of human diseases. To gain a better understanding of mitochondrial function, several recent proteomic, genetic, transcriptomic and bioinformatic approaches have set out to determine the complete set of mitochondrially located proteins in yeast, plants and mammals. Here, we review these studies and discuss the advances and limitations of individual strategies. Integration of various approaches proves to be a successful and useful way to identify the mitochondrial proteome with high sensitivity and specificity. The most comprehensive dataset is available for Saccharomyces cerevisiae, giving an estimated number of 700 different proteins located in mitochondria.