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For the first time, an HPLC–MS/MS method for the determination pmol/l levels of troventol (TRV) and clenbuterol as an internal standard (IS) in human plasma was developed, validated and tested on biological samples. The method included solid phase extraction by Waters Oasis WCX cartridges and chromatographic separation on a YMC-Pack SIL (100 mm × 2.1 mm, 5 μm, 12 nm) analytical column with acetonitrile–water–formic acid (50:50:0.1, v/v/v) as the mobile phase; the selected ion transitions were m/z 332.2→138.2 and m/z 277.0→203.1 for TRV and IS, respectively, in positive ionization mode. The calibration curve for TRV showed good linearity in the concentration range of 35–500 pg/ml. The method was applied to real samples taken from healthy subjects after inhalation of an aerosol containing 640 μg of TRV.