Objective?To establish an accurate，reliable method for determination of Isoniazid（INH）and Rifampicin（RFP）in human plasma by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS／MS），and investigate the pharmacokinetics characteristics in healthy Chinese male volunteers.Methods?By using stable isotopes as internal standards，the plasma samples were pretreated by precipitation with methanol，and then diluted and analyzed.Gradient elution was performed for both INH and RFP.INH was separated on an Atlantis HILIC by a HILIC Silica（50 mm×2.1 mm，3 μm）with 5 mmol／L ammonium formate，90% acetonitrile aqueous solution containing 0.01% formic acid as mobile phase.RFP were separated by a Syncronis C18（50 mm×2.1 mm，5 μm）with acetonitrile-50% acetonitrile aqueous solution as mobile phase.Electrospray ionization source was applied for determination in positive ion multi-reaction monitoring mode.Results?The linear ranges of INH and RFP were 5 to 5 000 ng／mL and 10 to 10 000 ng／mL respectively with fine correlation（r＞0.99）.The coefficient of variation of precision was less than 7%.No obvious matrix effect was found.Conclusion?The established method should be simple，accurate and specific and may be used in the study for human pharmacokinetics and drug monitoring in therapy.