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In this study a sensitive UHPLC-QTOF-MS method was developed and validated for the quantitation of the metformin (MET) and teneligliptin (TEN) in rat plasma using dapagliflozin as an internal standard (IS). Chromatographic separation were carried out on a Acquity UPLC HSS Cyano column (100 mm x 2.1 mm, 1.8 μm) using gradient mobile phase system consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.4 mL/min, within a run time of 6 min. Protein precipitation method was used as sample preparation approach. Detection of target ions [M+H]+ at m/z 130.1085 for MET, m/z 427.2277 for TEN and m/z 409.1623 for IS was performed at positive ion electrospray ionization mode. Linearity was assessed in the range of 0.98–1000 ng/mL for both MET and TEN. The developed assay was validated as per US-FDA and EMA bioanalytical guidelines and successfully applied to pharmacokinetic interaction study in SD rats. A 1.1 fold increment in the AUC levels of MET and TEN was observed when co-administered together in rats.First UHPLC-QTOF-MS method for the simultaneous quantification of metformin (MET) and teneligliptin (TEN) from rat plasma.Method was validated as per US-FDA and EMA bioanaytical guidelines.The method was specific, precise, accurate and linear from 0.98 to 1000 ng/mL.Application to pharmacokinetics and drug interaction studies in SD rats.