To support the clinical pharmacokinetic trials of bencycloquidium bromide (BCQB), a specific and sensitive method based on weak cation-exchange solid phase extraction (WCX-SPE) and HPLC-ESI-MS techniques for the determination of BCQB in human plasma was developed and validated. BCQB and the internal standard 1-ethyl-bencycloquidium bromide were separated on a Zorbax Eclipse Plus C18 column (3.5microm, 150mmx2.1mm i.d.) with a mobile phase consisted of 20mM ammonium acetate buffer solution containing 1% acetic acid (pH 3.6)-methanol (50:50, v/v), and the chromatographic run time for one sample was 8min. The lower limit of quantitation (LLOQ) of the method is 5pg/ml, which is critically important for the pharmacokinetic study of BCQB. The method possesses a reliable quantification range of 5-1000pg/ml, the acceptable intra- and inter-batch precision of less than 8.9%, and the extraction recovery of more than 90.6%. The method was successfully applied for the single-dose pharmacokinetic study of BCQB nasal spray in healthy Chinese volunteers.