Paeoniae Radix, a traditional herbal medicine, has been used in antispasmodic action, alleviating pain, and activating circulation. The aim of this study was to evaluate the pharmacokinetics (PKs) of paeoniflorin in healthy Korean subjects and develop an analytical method for the quantification of paeoniflorin in human plasma. This was an open-label, randomized, and single-dose study in 10 healthy Korean subjects who received a Paeoniae Radix extract powder. The plasma concentration of paeoniflorin up to 12 h was determined using a validated ultra-performance liquid chromatography tandem mass spectrometric method. The PK parameters such as AUC0-?, CL/F, Vd/F, Cmax, Tmax and t1/2 were calculated using WinNonlin_ software (version 6.4, Pharsight_, a CertaraTM Company). The best PK model of paeoniflorin in humans was the onecompartment model. The mean parameters were 2625.71 L/h, 10,150.55 L, 6.97 ng/mL, and 1.64 h for the oral clearance (CL/F), the volume of distribution (Vd/F), the maximum plasma concentration (Cmax), and the time to reach Cmax(Tmax), respectively.