Tinospora cordifolia (TC) has been used as a complimentary/alternative medicine against diabetes. Considering its potential to modulate metabolic enzymes, Tinospora cordifolia extract (TCE) may influence the metabolism of the antidiabeic drug Glibenclamide following co-administration. Accordingly, this work was undertaken to evaluate impact of TCE on fate of Glibenclamide. Activity of clinically important Cytochrome P450 isoenzymes were inhibited in the order of CYP2C9 > CYP2D6 > CYP2C19 > CYP1A2 > CYP3A4. Formations of metabolites were inhibited with increasing concentration of TCE in both rat and human liver microsomes. TCE was co- administered in three different groups (0, 100 and 400 mg/kg) with Glibenclamide at 1 mg/kg dose to observe the alteration in pharmacokinetic parameters of Glibenclamide. The rats were pretreated with 0 (vehicle), 100 and 400 mg/kg dose of TCE b.i.d for 14 days and on the 14th day all three groups were administered with 1 mg/kg Glibenclamide. Pharmacokinetic parameters were analyzed based on plasma concentrations of Glibenclamide from all the groups by LC–HRMS methods using Glipizide as an internal standard. At 400 mg/kg dose, a marked increase in the bio availability of Glibenclamide was observed with a significant delay of Tmax and suppression of clearance.