Gastrodin (Gas) and puerarin (Pur) are bioactive substances derived from traditional Chinese medicine Gastrodia elata and Radix Puerariae, respectively, which were often used together in Chinese clinical prescriptions. Their injections were used in combined way for treatment of some cardiocerebrovascular diseases in clinic, especially for vertigo due to vertebrobasilar ischemia. In this paper, interaction of gastrodin and puerarin in rat plasma pharmacokinetics via intragastic (i.g.)/intravenous (i.v.) administration was investigated. A reliable HPLC method was developed for simultaneous determination of Gas and Pur in rat plasma with a linear range of 0.101–101μg/mL for Gas and 0.0500–5.98μg/mL for Pur (r2>0.993). The LLOQ, LOD of Gas and Pur were determined to be 0.101, 0.0486μg/mL, and 0.05, 0.0245μg/mL, respectively. The intra-day and inter-day precision were all less than 12.0%, whilst the accuracy were all within 96.4±6.00%. The proposed method has been successfully applied to the pharmacokinetic study of the analytes in rats after i.g./i.v. administration of Gas and Pur alone or combined with each other (i.g.: 40mg/kg Gas, 400mg/kg Pur; i.v.: 20mg/kg Gas, 20mg/kg Pur). Blood samples were collected from retinal vein plexus of rats at predetermined time points and plasma containing the internal standard tyrosol (IS) were precipitated by methanol and chromatography was carried out on a C18 column with a gradient mobile phase of ACN–H2O with 0.05% phosphoric acid as a modifier. The pharmacokinetic profiles of combined administration were found to be distinct from those of given alone. The Cmax, Tmax, T1/2, MRT of Gas administrated alone or combined with Pur via i.g. were 21.7μg/mL, 0.250h, 2.81h, 0.830h and 18.4μg/mL, 0.550h, 0.970h, 1.37h, respectively, of Pur administrated alone or combined with Gas via i.g. were 0.490μg/mL, 1.95h, 1.33h, 2.10h and 2.01μg/mL, 0.570h, 4.00h, 5.10h, respectively. The relative oral bioavailability of Pur in combined administration was 10.7 times as much as that of single administration, whilst 1.52 folds in Gas. These results indicate that co-administration of Gas and Pur is a promising combination to gain higher bioavailability and it is suggested that doctors pay more attention to the dosages of the two when simultaneously using both of them.