Abstract: OBJECTIVE To establish an HPLC-MS/MS method for determination of the concentration of pyridostigmine bromide in human plasma and to study its application on pharmacokinetic. METHODS A liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and pharmacokinetic study of pyridostigmine bromide in human plasma using neostigmine methylsulfate as internal standard (IS). The chromatographic separation was performed on a SHIMADZU VP-ODS(150 mm×2.0 mm, 5 μm) analytical column using a mobile phase consisting of methanol and water containing 5 mmol·L^-1 ammonium acetate and 0.1% formic acid with gradient elution at a ?ow rate of 0.2 mL·min^-1. The detection was carried out on a triple quadrupole tandem mass spectrometer by selective reaction monitoring (SRM) via electrospray ionization interface with positive mode, monitoring the transition of the molecular ions m/z 181.1→72.2 for PB and 233.1→208.1 for IS. RESULTS Validation of the method demonstrated that the linear calibration curves covered the range of 0.5-100 ng·mL^-1, and the correlation coefficient was 0.996 9. The method was proved to be sensitive, specific, precise, accurate, and robust, revealing that it was appropriate for determination of pyridostigmine bromide in human plasma and successfully applied to the pharmacokinetics study after an oral administration of 60 mg pyridostigmine bromide tablets to twenty Chinese healthy volunteers. The mean of C_max and T_max were (43.26±10.33)ng·mL^-1 and (1.77±0.48)h, respectively. Plasma concentration declined with a t_1/2 of (5.11±0.96)h. AUC0?24 and AUC_0?∞ values obtained were (204.8±64.93)ng·h·mL^-1 and (212.42± 68.31)ng·h·mL^-1, respectively. CONCLUSION This paper describes a sensitive, specific and robust HPLC-MS/MS method for the determination of pyridostigmine bromide in human plasma. The method offeres a simple, economic, rapid and efficient extraction procedure, which is successfully applied to pharmacokinetic studies. The pharmacokinetic parameters in this paper can provide a theoretical basis for clinical medication.