(22R,S)Budesonide was isolated from human plasma by solid-phase extraction. Switching from reversed-phase conditions during sample application and washing to normal-phase conditions during elution afforded a very clean extract. Budesonide was derivatized with acetic anhydride to form the 21-acetyl derivative before analysis by reversed-phase liquid chromatography combined with thermospray mass spectrometry. Deuterium-labelled bude-sonide was used as internal standard. Standard samples prepared in human albumin solution were used for the calibration curve. An automated liquid chromatography/mass spectrometry system, allowing unattended overnight operation, was used for routine analysis. The recovery of budesonide from plasma was 88.9 ± 5.9% (mean ± SD) and the method was linear over the range 0.30–30 pmol (amount analysed), corresponding to plasma concentrations of 0.10–10 nmol l−1. Budesonide could be measured down to 0.10 nmol l−1 with a within-day variation of 10–18% (CV). The error was less than ± 15% at 0.10 nmol l−1 and less than ± 7% at concentrations of 0.20 nmol l−1 or higher. The total imprecision between days was 9% (CV) at a concentration of 0.30 nmol l−1.