In this report, a high-performance liquid chromatography-electrospray ionization mass spectrometric (HPLC-MS) method was used to determine gastrodin (GAS) and p-hydroxybenzylalcohol (HBA) in rat plasma after oral administration of Gastrodia elata Bl. (Chinese name: Tianma) extract. Up to 200 microl of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the concentration range tested 2.00-200.00 microg/ml for GAS and 0.832-104.00 microg/ml for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83 microg/ml for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 91.12-108.64% with RSD less than 7.80% for GAS and 92.91-106.14% with RSD less than 4.30% for HBA. Evidence showed that a rapid, simple and reproducible LC-MS assay was established to determine GAS and HBA in rat plasma.