A new liquid chromatography tandem mass spectrometric method for simultaneous estimation of anti-hyperglycemic agents, metformin, linagliptin, sitagliptin and vildagliptin from human plasma was developed and validated as per ICH/USFDA guidelines. Chromatographic separations were achieved using Chromolith^® High Resolution RP-18e HPLC column (100 mm × 4.6 mm, macropores 1.15 μm) with an isocratic elution mode using the mobile phase composed of 0.01 M ammonium formate buffer (pH 3.0): acetonitrile (80:20 v/v). A flow rate of 0.4 mL min⁻¹ was maintained throughout the analysis. Detection was performed by triple quadrupole MS fitted with ESI probe functioning in the positive ion MRM mode. Extractions of all the drugs from plasma were carried out by acetonitrile crash technique. Alogliptin was used as an internal standard to minimize the error which could occur during analysis. The validation study data demonstrated that the new method is highly selective and sensitive (the limits of detection were 1.76, 1.94, 0.17 and 3.08 ng mL⁻¹ for metformin, linagliptin, sitagliptin and vildagliptin, respectively). The %CV and %RE values were within the acceptable limit, <1% for most of the data, which indicate that the reported method was very precise and accurate. A linear calibration curve (correlation coefficient, r ² > 0.999) was obtained at the concentration range of 0.5–400.0; 5.0–400.0; 10.0–500.0 and 0.5–40.0 ng mL⁻¹ for metformin, linagliptin, sitagliptin and vildagliptin, respectively. The extraction efficacy was evidenced from recovery study and all four analytes were found to be stable in plasma. The developed LC–MS/MS method is robust and can be applied for monitoring plasma levels of analyzed antidiabetic drugs in preclinical and clinical pharmacokinetic studies.