Abstract A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid–liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C 8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/ z 631.8 → 373.1, m/ z 393.2 → 147.1 and m/ z 264.2 → 58.1, respectively. The method was linear over the ranges 1.5–1000 ng/mL for dexamethasone palmitate and 2.5–250 ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100 ± 7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.