The determination of the synthetic corticosteroid dexamethasone in equine urine is described using coupled on-line immunoaffinity chromatographyâ high-performance liquid chromatography (IACâ HPLC) with detection by UV or mass spectrometry. The limit of detection for dexamethasone with UV detection was 30 µg l^{â 1} for a 10 ml urine sample. Atmospheric pressure chemical ionization mass spectrometry, with single ion monitoring, gave a limit of detection of 0.1 µg l^{â 1} for dexamethasone. The determination of dexamethasone by on-line IACâ HPLCâ MS has been demonstrated in post-administration equine urine samples.