Background: Pharmacologic treatments for type 2 diabetes are based upon increasing insulin availability and improving sensitivity to insulin. Nowadays, glucagon like peptide-1 (GLP-1) based therapies aims at glucose control through DPP-4 inhibitors. DPP-4 is a transmembrane glycoprotein belongs to prolyl oligopeptidase family, with the specificity of removing X-Pro or X-Ala dipeptides from the N-terminus of polypeptides. GLP-1 effect by stimulating glucose-dependent insulin release from the pancreatic islets, inhibit inappropriate post-meal glucagon release and slow gastric emptying promoting leaky gut. The current study investigated DPP-4 inhibitory activity of catechin, isolated from Withania somnifera (WS), for ethnopharmacological treatment of type 2 diabetes and aimed to increase availability of GLP-1and sensitivity to insulin. Materials and Methods: Young and matured fresh roots, leaves, and fruits of WS plant extract were considered and were systematically evaluated for DPP-4 inhibitory activity using in vitro method, enzyme kinetics, phytochemical analysis, RP-HPLC, LCMS and 1 H and 13 C NMR method and structure-activity relationship (SAR) studies. Results: In this study, methanol (100% and 80%) extracts of WS matured root exhibited maximum DPP-4 inhibitory activity when compared to other extracts. The maximum DPP-4 inhibitory activity was found in 100% methanol extract of matured root. Phytobioactive was purified by RP-HPLC. The compound purified was found to be flavonoid and was characterized (LCMS, 1 H and 13 C NMR studies), identified as catechin. Auxiliary, molecular docking was performed using Ligand Fit method using PatchDock package. The study revealed the binding affinity of catechin with DPP-4 to be -6.601 kcal/mol with 13 hydrogen interactions with the receptor and was very similar to the standard potent blockers withaferin A and others (cuscohygrine, scopoletin, sitoindoside IV, tropine), further confirming its hyperglycemic potency. Conclusion:The study reveals that, 100% methanol extract of WS matured roots contains the compound-catechin, which exhibits DPP-4 inhibitory activity resulting in increased level of bioactive GLP-1 and GIP. In this background, we concluded that the WS will be a better source for further development as new antidiabetic drugs.