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Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was used for simultaneous quantification of tolterodine and its metabolite 5-hydroxy methyl tolterodine in rat plasma. Tolterodine-d6 and 5-hydroxy methyl tolterodine-d14 were used as internal standards (IS). Chromatographic separation was performed on Ascentis Express RP amide (50 mm×4.6 mm, 2.7 μm) column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile in the ratio of 20:80 (v/v), at a flow-rate of 0.5 mL/min. Tolterodine, tolterodine-d6, 5-hydroxy methyl tolterodine and 5-hydroxy methyl tolterodine-d14 were detected with proton adducts at m/z 326.1→147.1, 332.3→153.1, 342.2→223.1 and 356.2→223.1 in multiple reaction monitoring (MRM) positive mode respectively. The drug, metabolite and internal standards were extracted by liquid–liquid extraction method. The method was validated …