Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins.