The study investigates the direct activation of SIRT1 by SRT1720, SRT2183, SRT1460, and resveratrol using biochemical assays with native peptide and full-length protein substrates. The results show that these compounds do not activate SIRT1 with native peptide or full-length protein substrates but activate SIRT1 with peptide substrates containing a covalently attached fluorophore. Biophysical studies, including NMR, surface plasmon resonance, and isothermal calorimetry, provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Additionally, SRT1720 does not lower plasma glucose or improve mitochondrial capacity in mice fed a high-fat diet. The compounds exhibit off-target activities against various receptors, enzymes, transporters, and ion channels. Therefore, the study concludes that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.The study investigates the direct activation of SIRT1 by SRT1720, SRT2183, SRT1460, and resveratrol using biochemical assays with native peptide and full-length protein substrates. The results show that these compounds do not activate SIRT1 with native peptide or full-length protein substrates but activate SIRT1 with peptide substrates containing a covalently attached fluorophore. Biophysical studies, including NMR, surface plasmon resonance, and isothermal calorimetry, provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Additionally, SRT1720 does not lower plasma glucose or improve mitochondrial capacity in mice fed a high-fat diet. The compounds exhibit off-target activities against various receptors, enzymes, transporters, and ion channels. Therefore, the study concludes that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.