The SIRT-Glo Assay is a single-reagent-addition, homogeneous, luminescent assay that measures the relative activity of the NAD+-dependent histone deacetylase (HDAC) class III enzymes (sirtuins; SIRTs) from purified enzyme sources. The assay uses an acetylated, luminogenic peptide substrate that can be deacetylated by SIRT activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo recombinant firefly luciferase. The assay reaction is typically complete within 15-45 minutes with no sample manipulation. The SIRT-mediated luminescent signal is persistent with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The SIRT-Glo Assay is broadly useful for NAD+-dependent Sirtuin enzymes. Nicotinamide, available separately, is a known inhibitor of SIRTs and used as a positive control inhibitor. Nicotinamide is supplied at a concentration of 1M in SIRT-Glo Buffer. The HeLa Nuclear Extract, available separately, may be used as an assay chemistry control. HeLa Nuclear Extract is supplied at a concentration of 5mg/ml.