Sirtuins refer to a family of intracellular enzymes that are the yeast silent information regulator 2 (sir2) protein homologs found in organisms from all the three kingdoms of life. This family of enzymes primarily catalyze the protein Nɛ-acyl-lysine deacylation reaction despite the report for a type of bacterial/fungal sirtuins to robustly catalyze a protein mono-ADP-ribosylation reaction, however, these two group transfer reactions employ the redox coenzyme β-nicotinamide adenine dinucleotide (β-NAD+) as the obligatory cosubstrate. Since 2000, in addition to histone proteins, more and more nonhistone proteins have also been identified as native substrates for the sirtuin-catalyzed deacylation, consistent with the ever-increased demonstration that this enzymatic reaction plays an important regulatory role in a variety of cellular processes, such as gene transcription and metabolism. This latter role is also consistent with the absolute dependence on β-NAD+ of the deacylation reaction catalyzed by sirtuin family members. The sirtuin-catalyzed deacylation has further been proposed as a contemporary therapeutic target for human diseases, such as cancer, neurodegenerative and metabolic diseases. In order to fully tap the therapeutic potential of the sirtuin-catalyzed deacylation, the past few years have witnessed a tremendous advancement in mechanistic elucidation, chemical modulator (inhibitor and activator) development, (chemical) biological and pharmacological exploration of the sirtuin-catalyzed deacylation reaction. During the journey of this knowledge advancement, the use of carefully designed chemical probes has played an elegant role. This chapter will delineate the development and application of these chemical probes in sirtuin research.