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Computer Aided Drug Design (CADD) molecular docking program used in this study and as a result Rifabutin was retrieved as structurally similar drug molecule to Lixisenatide (A widely used GLP-1 agonist). To afford beneficial reason in vitro study conducted which too provides a favourable mark for prospective inhibition of α-glucosidase and α-amylase enzyme and thereby evaluated for its antidiabetic potential against Streptozotocin (STZ) and Nicotinamide (NA) induced diabetes in albino wistar rats. Rifabutin demonstrated significant efficacy towards restoring the blood glucose, insulin and distorted lipid profile in the animals treated with STZ-NA. Rifabutin also regulated the inflammatory (Nitric Oxide) and oxidative stress (TBARs, SOD, Catalase, GSH and protein carbonyl) markers in the pancreatic tissue of the STZ-NA treated animals. When scrutinized on the grounds electrocardiographic and heart rate variability (HRV) changes, Rifabutin decreased the heart rate, without much affecting the QRS, QT and QTc interval. Rifabutin was also evident to be less hepatotoxic in comparison to the standard metformin, providing an added advantage as an anti-diabetic agent. The study concluded that the low dose of rifabutin can favourably regulate of the blood glucose and other biochemical paradigms in comparison to high dose of rifabutin.