Inhibitors of human NAD(+)- dependent protein deacetylases possess great value for deciphering the biology of these enzymes and as potential therapeutics for metabolic and age-related diseases and cancer. In the current study, we have experimentally demonstrated that, the potent inhibition we obtained previously for one of these enzymes (i.e. sirtuin type 1 (SIRT1)) by simply replacing N-epsilon-thioacetyl-lysine for N-epsilon-acetyl-lysine in its peptide substrate, represented a general and efficient strategy to develop potent and selective inhibitors of human NAD+- dependent protein deacetylase enzymes. Indeed, by using this simple inhibition strategy, potent (low-micromolar) and selective (<= 40-fold) SIRT2 and SIRT3 inhibitors, which were either comparable or superior to currently existing inhibitors, have also been quickly identified in the current study. These inhibitors could be used as chemical biological tools or as lead compounds for further focused structure-activity optimization.