Histone deacetylase (HDAC) family of enzymes catalyze the specific lysine N(epsilon)-deacetylation on proteins such as the core histone proteins, various transcription factors, alpha-tubulin, and acetyl-coenzyme A synthetase that are respectively involved in gene transcriptional, cytoskeletal, and metabolic control. HDAC-catalyzed reaction represents an integral component for an emerging intracellular signaling mechanism defined by the protein posttranslational reversible lysine N(epsilon)-acetylation and deacetylation, and has been targeted for developing novel therapies for metabolic and age-related diseases and cancer. In this study, we disclose the synthesis and characterization of N(epsilon)-thioacetyl-lysine as a multi-facet tool for enzymatic protein lysine N(epsilon)-deacetylation. In specific, by evaluating multiple series of peptides containing N(epsilon)-thioacetyl-lysine, i) we developed the first spectrophotometric assay selective for HDAC8 that holds potentials for not only inhibitor screening but also selective activity reporting for HDAC8 activity; ii) we identified potent and selective peptide-based inhibitors for SIRT1, SIRT2, and SIRT3 that are the only bona fide human class III protein deacetylase enzymes with known physiological substrates. All these results have far-reaching impact on promoting our fundamental understanding and pharmacological exploitation of HDAC-catalyzed reactions.