A rapid, sensitive and specific method to determination of ambroxol in human plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC–MS/ESI) was described. Ambroxol and the internal standard (I.S.), fentanyl, were extracted from plasma by N-hexane-diethyl ether (1:1, v/v) after alkalinized with ammonia water. A centrifuged upper layer was then evaporated and reconstituted with 100 μl mobile phase. Chromatographic separation was performed on a BDS HYPERSIL C18 column (250 mm × 4.6 mm, 5.0 μm, Thermo electron corporation, USA) with the mobile phase consisting of 30 mM ammonium acetate (0.4% formic acid)–acetonitrile (64:36, v/v) at a flow-rate of 1.2 mL min−1. The total run time was 5.8 min for each sample. Detection and quantitation was performed by the mass spectrometer using selected ion monitoring at m/z 261.9, 263.8 and 265.9 for ambroxol and m/z 337.3 for fentanyl. The calibration curve was linear within the concentration range of 1.0–100.0 ng mL−1 (r = 0.9996). The limit of quantification was 1.0 ng mL−1. The extraction recovery was above 83.3%. The methodology recovery was higher than 93.8%. The intra- and inter-day precisions were less than 6.0%. The method is accurate, sensitive and simple for the study of the pharmacokinetics and metabolism of ambroxol.