A simple and precise reversed-phase high performance liquid chromatography (HPLC) assay for the determination of dexamethasone in human plasma was developed and validated. 1.0 ml plasma samples containing dexamethasone and lansoprazole as internal standard (IS) were deproteinized with 2 ml dichloromethane and 3 ml methyl tert-butyl ether. Separation was achieved on Atlantis dC-18 column with a mobile phase consisted of acetonitrile and water (40:60, v:v), and delivered at a flow rate of 1.0 ml/min. The eluent was monitored spectrophotometrically at 240 nm. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of dexamethasone in plasma and peak height ratio of dexamethasone to the IS was linear over the range of 0.15-8.0 μg/ml. Intra-day and inter-day coefficient of variation (CV) and bias were ≤11.1% and ≤9.5% and ≤9.3% and ≤5.6%, respectively. Mean extraction recovery of dexamethasone and IS was 100% and 97%, respectively. The method was applied to assess dexamethasone stability under various conditions encountered in the clinical laboratory. Dexamethasone stability in processed (24hrs at room temperature, 48hrs at -20°C) and unprocessed (24hrs at room temperature, 8 weeks at -20°C, or after 3 cycles of freeze and thaw) samples was ≥ 90% and ≥ 83%, respectively