Sirtuins (SIRTs) are a family of NAD⁺-dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age-related metabolic diseases and cancers, so SIRT modulators are considered to have potential therapeutic values. For a long time, it was thought that SIRTs only catalyze deacetylation reactions of histones, however, more recently, it was found that SIRTs can remove various acyl groups, including propionyl, crotonyl, succinyl, hexanoyl, myristoyl, palmitoyl, lipoyl and benzoyl groups, from histones and many other protein substrates. In addition, recent reports suggest that SIRT6 demyristoylase activity regulates the secretion of TNF-α, and that SIRT2 defatty acylase activity regulates the localization of K-Ras4a oncoprotein and promotes cellular transformation, meaning that new enzymatic activities of SIRTs also regulate pathophysiological functions. Therefore, SIRT-subtype- and activity-specific fluorescence probes and modulators are required both as tools for basic research to understand the physiological functions of SIRTs and as candidate therapeutic agents. In this review article, we first describe the molecular design and development of a new fluorescent probe for one-step SIRT activity detection and their application to cellular SIRT1 imaging. We then outline a strategy for converting fluorescent probes to peptide inhibitors based on the findings obtained in the course of developing the fluorescent probes. We further describe the construction of a fluorescent probe library with different isozyme selectivity among SIRTs was used to select the best fluorescent probe for detecting SIRT2 activity, which was then applied to chemical screening to search for small-molecular inhibitors of SIRT2 demyristoylase activity.