Sirtuins are nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylases that mediate cellular processes such as lifespan extension and metabolic regulation. Sirtuins form a unique metabolite, 2'-O-acetyl-ADP-ribose (OAADPr), shown to block oocyte maturation, bind to chromatin-related proteins, and activate ion channels. Given the various sirtuin phenotypes, the potential of OAADPr as a signaling molecule is extensive. However, exploration of the biological roles of OAADPr has been hindered by the lack of in vivo evidence and a reliable method for quantification. Here we provide the first direct evidence and quantification of cellular OAADPr. Compared with endogenous OAADPr levels (0.56+/-0.13 microM) in wild-type Saccharomyces cerevisiae, deletion of all five yeast sirtuins (Sir2 and Hst1-4) yielded essentially no detectable OAADPr. The single deletion of Hst2 yielded 0.37+/-0.12 microM OAADPr. Deletion of an enzyme, Ysa1, previously shown in vitro to hydrolyze OAADPr, resulted in a significant increase (0.85+/-0.24 microM) in OAADPr. Together, these data provide evidence that cellular levels of OAADPr are controlled by the action of sirtuins and can be modulated by the Nudix hydrolase Ysa1. Our methodology, consisting of internal standard (13)C-labeled OAADPr and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, displays excellent sensitivity and a linear dynamic range from 0.2 to 500 pmol. Moreover, extraction efficiencies were greater than 75%. This methodology is an essential tool in probing the biological roles of OAADPr, especially under conditions in which sirtuin phenotypes are well established.