Sirtuins belong to the class III histone deacetylase family with NAD-dependent protein lysine deacylase activities, which has been shown to play important roles in many physiological processes, including the regulation of transcription, DNA repair, metabolism and protein secretion. Small molecules that can modulate Sirtuin activity are important for fundamental studies of Sirtuin biology and drug development. High-throughput assays to screen for small molecule modulators of Sirtuins have been approved to be very useful tools for the development of therapeutics and research tools targeting Sirtuins. In this review, we will summarize the reported assays (during 2000-2015) for Sirtuins and discuss how these assays show different performances for the possibility of screening Sirtuin modulators and the application of high-throughput format screening. (C) 2015 Elsevier Inc. All rights reserved.