Owing to its presence in several biological processes Sirt1 served as a potential therapeutic target for many diseases. Here we report the synthesis of two distinct series of novel Sirt1 selective inhibitors, benzimidazole monopeptides and 5-pyrazolyl methylidene rhodanine carboxylic acid derived amino acids, constructed using structure-guided computational approaches. Furthermore, compounds were evaluated, against human Sirt1-3 for in-vitro inhibitory activity compared to Ex527 (reported Sirt1-selective inhibitor), in liver and breast cancer cell lines for cytotoxicity. The tryptophan conjugates 13h (IC50 = 0.66 µM) and 7d (IC50 = 0.77 µM) demonstrated maximum efficacy to inhibit Sirt1. Molecular dynamics simulations unveil the interaction map and electrostatic complementarity at substrate binding site, could be a cause of selective Sirt1 inhibition. Furthermore, the Sirt1 inhibition was monitored via increased p53 acetylation status checked in HepG2 cells. These findings will pave the pathway for developing novel selective Sirt1-inhibitors in cancer therapeutics.