A new simple, rapid and sensitive high pressure liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for simultaneous analysis of mebeverine metabolites as: mebeverine alcohol (MAL), mebeverine acid (MAC) and desmethylmebeverine acid (DMAC) in human plasma. Sample preparation was performed by protein precipitation following the separation of analytes using an Acquity UPLC BEN C8 column 1.7 mm 2.1 × 50 mm (Waters, USA). 2H5-desmethylmebeverine acid (2H5-DMAC) was used as the internal standard (IS). The proposed method was validated with linear ranges of 0.1–10 ng/mL; 1–100 ng/mL and 5–1000 ng/mL for MAL, MAC and DMAC, respectively. Accuracy for all analytes (%RE), given as deviation between nominal and measured concentration and assay variability (CV) ranged from −4.04% to 4.60% and from 0.31% to 6.43% respectively both for within- and between-run. The overall recoveries for all metabolites were above 85%. The proposed method was used successfully for analysis of real samples from a pharmacokinetics study.