The sirtuins represent a distinct class of trichostatin A-insensitive lysyl-deacetylases (class III HDACs) that catalyze a reaction coupling lysine deacetylation to the formation of nicotinamide and O-acetyl-ADP-ribose. The SIRT2 Direct Fluorescent Screening Assay provides a fluorescence-based method for screening SIRT2 inhibitors or activators. The procedure requires only two easy steps, both performed in the same microplate. In the first step, the substrate, which comprises the p53 sequence Gln-Pro-Lys-Lys(ε-acetyl)-AMC, is incubated with human recombinant SIRT2 along with its cosubstrate NAD+. Deacetylation sensitizes the substrate such that treatment with the Developer in the second step releases a fluorescent product.