Sirtuins (SIRTs) form a critical family of nicotinamide adenine dinucleotide (NAD)-dependent enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, SIRTs4-7 have little to no deacetylase activity, and a robust catalytic activity for mitochondrial SIRT4 has remained elusive. Moreover, in vitro characterization of SIRT4 has been hampered by difficulty in maintaining soluble and active recombinant protein. Therefore, to investigate potential cellular substrates of SIRT4, we used proteomics to define its mitochondrial protein interactions in human fibroblasts.