Together with other post-translational modifications, acylation of proteins is a powerful means of regulation of their activity. Some acylation types occur nonenzymatically and are driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein as it changes the charge of the residue. In eukaryotes it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-coenzyme A, is primarily generated in the tricarboxylic acid (TCA) cycle. Although numerous succinylated mitochondrial proteins were identified in Saccharomyces cerevisiae, a detailed characterization of yeast mitochondrial succinylome is lacking. Here we performed a proteomic mass spectrometry analysis of purified yeast mitochondria and detected 313 succinylated mitochondrial proteins with 1762 novel succinylation sites.