Abstract: OBJECTIVE To establish an UPLC-MS/MS method for the determination of the concentrations of cyperenone and α-cyperone in rat plasma and to study the pharmacokinetics. METHODS Gradient elution was carried out on a Phenomennex C₁₈(150 mm×2.0 mm, 3 μm) column with acetonitrile-water as mobile phase. The column temperature was 30 ℃, injection volume was 1 μL, osthenite was used as the internal standard, electrospray ion source and positive ion mode were used. The m/z values of cyperenone, α-cyperone and osthenite were 219.1/135.1, 219.1/111.0 and 245.0/123.0, respectively. The plasma concentrations of cyperenone and α-cyperone were measured, and the main pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS The linear relationship of cyperenone was good in the range of 10-500 ng·mL^-1(r=0.991 0), and the linear relationship of α-cyperone was good in the range of 2.5-300 ng·mL^-1(r=0.994 1), RSDs of intra-day precision were less than 9.45%. RSDs of daytime precision were less than 9.09%. The recoveries were greater than 86.79%. After intragastric administration of essential oil extract(20 mg·kg^-1) from Cyperus rotundus L. in SD rats. The pharmacokinetic parameters of C_max, AUC_0-∞ and MRT_(0-∞) of cyperenone and α-cyperone were (8 862.59±1 106.81)ng·L^-1, (7 060.94±774.25)ng·L^-1·h, (3.21±0.72)h and (934.69±106.81)ng·L^-1, (792.26±74.52)ng·L^-1·h, (4.94± 0.82)h, respectively. CONCLUSION The established method can be used for the rapid and accurate determination of the concentration of cyperenone and α-cyperone in plasma, and can be used for the pharmacokinetic study of cyperenone and α-cyperone in rats in vivo.