Abstract: Mitiglinide in human plasma was determined by LC-MS/MS. Mitiglinide and the internal standard were extracted from plasma by acetonitrile, which was used as deproteinated solvent, and then separated on a Kromasil-C₁₈ column (50 mm×4.6 mm×5 μm). The mobile phase consisted of acetonitrile1 mmol•L^-1 ammonium formate (containing 0.1% formic acid) maintained at 30 ℃. The flow rate was 0.6 mL • min^-1, and 20 μL aliquot of residues were injected into the LC-MS/MS system. Detection was carried out by multiple reaction monitoring on an 3200Qtrap LC-MS/MS system. The assay is linear over the range 3.0-3 000.0 μg •L^-1 with a lower limit of quantitation of 3.0 μg •L^-1. Intra-day and inter-day precision are both less than 15%, respectively. The relative deviation is in the range of -1.40%-2.07%. The recoveries of Mitiglinide are (98.2±7.6)%, and stabilities are good. It is a rapid, sensitive, selective and reliable method for the determination of Mitiglinide in human plasma. The assay can be applied for the determination of Mitiglinide in human plasma and the study on pharmacokinetics.