A sensitive, rapid and simple liquid chromatographyelectrospray ionization mass spectrometry (LC-ESI-MS/MS) method was developed for the quantitative determination of cyclobenzaprine in human plasma, to study the pharmacokinetic behavior of cyclobenzaprine capsule in healthy Chinese volunteers. With escitalopram as the internal standard (IS), sample pretreatment involved a one-step liquidliquid extraction using saturated sodium carbonate solution and hexanediethyl ether (3:1, v/v). The separation was performed on an Ultimate XB-CN column (150?x?2.1?mm, 5?mu m). Isocratic elution was applied using acetonitrilewater (40:60, v/v) containing 10?m M ammonium acetate and 0.1% formic acid. The detection was carried out on a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. The ion-pairs including m/z 276.2216.2 for cyclobenzaprine and m/z 325.2109.1 for IS were used for monitoring. Linear calibration curves were obtained over the range of 0.04929.81?ng/mL with the lower limit of quantification at 0.049?ng/mL. The intra- and inter-day precision showed =6.5% relative standard deviation. The established method laid the groundwork for follow-up studies and provided basis for the clinical administration of cyclobenzaprine. Copyright (c) 2011 John Wiley & Sons, Ltd.