Abstract A high-performance liquid chromatography (LC–MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. An 8 mm disc was cut from the dried blood spot and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2 ml/min. LC–MS detection was conducted with single ion monitoring using target ions at m/ z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15–800 ng/ml. The overall extraction recovery of dexamethasone from dried blood spot samples was 99.3% (94.3–105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from a premature neonate. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30 μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.