In this paper the authors proposed a simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay method for the determination of darifenacin in human plasma. This method employed solifenacin as an internal standard (IS). Analyte and the IS were extracted form 200 µL of human plasma using protein precipitation technique. The chromatographic separation was achieved on a C18 column by using a mixture of acetonitrile and 5mM ammonium formate in 0.01% formic acid (90:10, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The linearity of the method was established in the concentration range 0.05–20.5 ng/mL with r2 ³ 0.99. The intra–day and inter–day precision (%CV) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. The validated method was successfully applied to a pharmacokinetic study in humans under fasting condition with 15 mg darifenacin extended release tablet.