To investigate the pharmacokinetic and bioavailability of polydatin (PD) in rats after oral and intravenous administration, a simple, rapid and sensitive liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method was developed and validated for the determination of polydation. After precipitating the plasma proteins with methanol, the analytes were separated on a C18 column (3.5µm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-acetonitrile-0.1% formic acid (18: 15: 67, v/v/v) at a flow rate of 0.3mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring (MRM) mode and the electrospray ionization technique was in negative mode. Linear responses were obtained for PD ranging from 1.0-5000.0 ng/mL (r= 0.9984) and the LLOQ was 1.0ng/ml and was sufficient for the pharmacokinetic studies. The intra-day and inter-day accuracy and precision of the assay were less than 8.0%. The method is capable of quantifying PD. The pharmacokinetic parameters of polydatin after intragastric administration of PD with different doses (50, 100 and 300 mg/kg) and intravenous administration at the dose of 20 mg/kg, were obtained, with t1/2 of 200.30 min, 210.30 min, 272.26 min, and 112.5 min, and AUC0-∞ of 125626.41 μg/L· min, 250433.47 μg/L·min, 693722.60 μg/L· min and 1723509.57μg/L· min, respectively. The absolute bioavailability of PD was somewhat low to 2.9%. The results were firsly reported, as far as we know, about bioavailability of PD and seem important for linking PD and other phenolic glycosides-related drugs administration to their medicinal effects.