Abstract Herbal medicines have been used from ancient times for the management of several disease and ailments along with prescribed medicine without consulting the physicians. Berberis aristata DC. has been widely used in management of diabetes as alternative therapy in India and other Asian countries. An approach has been made to evaluate the possible cytochrome P450 (CYP450) enzyme inhibition potential with the bark of the anti-diabetic herb Berberis aristata DC. along with combination of oral hypoglycaemic agents like glimepiride and gliclazide. Bioactive compound was quantified through RP-HPLC, in order to standardize the plant extract and interaction potential of standardized extract and bioactive compound with oral hypoglycaemic drugs were evaluated. Herb–drug interaction potential of the test samples were evaluated by CYP450-carbonmonoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human CYP450 isoforms such as CYP3A4, CYP2D6, CYP2C9 and CYP1A2 isozymes were analyzed through fluorescence microplate screening assay. The combination index (CI)—isobologram method was applied for the prediction of combination effects. Plant extract showed significantly higher IC50 value than respective positive control against CYP3A4, 2D6, 2C9 and 1A2. Combination of the test extract and the phytoconstituent present therein along with oral hypoglycemic drugs also showed significantly less (P