To establish a method for the determination of cucurbitacin in plasma samples, in order to study the in vivo pharmacokinetic characteristics of cucurbitacin in rats. Rats were intravenously injected with cucurbitacin. With diphenhydramine as the internal standard (IS), the plasma concentrations of cucurbitacin in rat plasma at different time points were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). With electrospray ionization source, the positive ion detection in the multiple reaction monitoring mode was conducted to determine the ion-pairs for target compound and IS were m/z 503.2/113.1 and m/z 256.0/167.2, respectively. Agilent ZOBAX SB-C18 column (2.1 mm x 50 mm, 1.8 microm) was adopted and eluted with methanol and 0.1% formic acid (55:45), and the flow rate was 0.2 mL x min(-1). DAS 2.0 software was applied to fit the blood concentration and calculate corresponding pharmacokinetic parameters. The rats were intravenously injected with cucurbitacin at the concentration of 3.0 mg x kg(-1). The target blood quality concentration show good linear relations within the range of 10.5-3 150 microg x L(-1) (R2 = 0.996), the lower limit of the standard curve was 10.5 microg x L(-1), and the signal to noise ratio S/N = 12. Intra- and inter-day precisions RSD was less than 6.9% and 14%, respectively; The accuracy RE ranged between 0.20% and 3.7%; The extraction recoveries ranged between 92.7% and 97.1%. Regarding the pharmacokinetic parameters of tail intravenous injection of cucurbitacin, AUC (0-t) was (811.615 +/- 111.578) microg x h x L(-1), (t1/2) was (1.285 +/- 1.390) h, CL was (3.627 +/- 0.487) L x h x kg(-1), and V(d) was (6.721 +/- 7.429) L x kg(-1). In this study, researchers established a simple, accurate, sensitive and highly specific method for determining the blood concentration of cucurbitacin, and reported the in vivo pharmacokinetic characteristics of cucurbitacin in rats for the first time.