In order to investigate the pharmacokinetics of a combination of diazepam and dexamethasone under hypoxic conditions, a novel, sensitive and specific liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of diazepam and dexamethasone in rat plasma was developed and validated. The chromatographic separation of analytes was successfully achieved on an XTerra® MS C18 column using a gradient elution of methanol and water containing 0.1% formic acid at a flow rate of 0.5 mL/min. This method demonstrated good linearity and no endogenous material interferences. The linear ranges were 1.0-100 ng/mL for diazepam and 2.0-200 ng/mL for dexamethasone. The intra- and inter-day precision for the two compounds in plasma were lower than 10.0%, and the accuracy was between -7.9% and 11.5%. Our method was then successfully applied in a pharmacokinetic comparison between normoxic and hypoxic rats. The results indicated that there were significant differences in the main pharmacokinetics parameters of diazepam and dexamethasone between normoxic and hypoxic rats. The results provide the important and valuable information for discovering and developing novel anti-hypoxia drug combinations, as well as a better understanding of the safety and efficacy of these drugs.