A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) assay for the determination of bencycloquidium bromide (BCQB) in rat plasma was firstly developed and validated. After addition of 1-ethyl-bencycloquidium bromide as an internal standard (I.S.), the plasma samples were deproteinized with methanol and the supernatant was assayed by LC-ESI-MS. Chromatographic separation was achieved with a Hanbon Lichrospher 5-C18 column. The mobile phase consisted of methanol-40 mM ammonium acetate buffer-formic acid (75:25:0.25, v/v/v) and delivered at the flow rate of 1.0 ml/min. LC-ESI-MS was carried out on a single quadrupole mass spectrometer using electrospray ionization (ESI) and positive selected-ion monitoring (SIM). Target ions were monitored at [M](+)m/z 330.2 for BCQB and [M] (+)m/z 344.2 for I.S. Calibration curve was linear over the range of 3-1500 ng/ml. The lower limit of quantification (LLOQ) was 3.0 ng/ml. The intra- and inter-run relative standard deviations (R.S.D.%) of the assay were less than 7.1 and 12.3%, respectively. The accuracy determined at the concentrations of 3.0, 100.0, 500.0 and 1500 ng/ml for BCQB were within +/-15.0%. The established method has been applied successfully to study the pharmacokinetics of BCQB in rats after intranasal administration.