Aim:To develop a sensitive and specific LC-MS method for the quantitative analysis of phenobarbital(PB) in rat hippocampus.Methods:PB was extracted from rat hippocampus by the mixed solvents of ether and cyclohexane after theophylline was added as internal standard.After evaporation,the residual was reconstituted with methanol and centrifuged.The upper aliquot was used for injection onto HPLC system.The chromatographic analyses were performed on a by Shim-pack ODS C_(18) column(150 mm×2.0 mm ID,5.0 μm) with the mobile phase which was operated in the gradient mode using mobile phase A(methanol-0.03% formic acid-0.004% triethylamine) and mobile phase B(methanol) at a flow rat of 0.2 mL/min.Detection was carried out on an electrospray ionization(ESI) mass spectrometer;operated in selected ion monitoring(SIM) and negative-ionization mode of target ion m/z 231 for PB and m/z 179 for the theophylline.Results: Under this analytic setting,there was good resolution between PB and IS.The calibration curve was linear in the range of 10～2000 ng/mL with r>0.99 and the limit of quantitation of 10 ng/mL.The recoveries of the analytes were more than 87%,and both intra-batch and inter-batch RSD were less than 10%.In addition,freeze-thaw stability,matrix effect,and quality control were with acceptable data. Conclusion:A sensitive,reliable and accurate method suitable for the quantitation of PB in rat hippocampus has been established.