A sensitive LC-ESI-MS method has been developed and validated for the determination of bencycloquidium bromide (BCQB) in human urine samples. The method utilized a solid-phase extraction (SPE) procedure, choosing carboxy propyl phase (CBA) as the extracting sorbent for purification of BCQB, with better baseline and higher selectivity achieved. Sample preparation by this method yielded very good and consistent mean recovery of above 94.5%. Another major benefit of the present method was the high detectability, with a lower limit of quantification (LLOQ) of 0.02ng/ml. The developed method was successfully applied to determine BCQB in human urine, and was proved to be suitable for use in Phase I clinical pharmacokinetic study of BCQB.