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Due to their marked antiinflammatory effect, synthetic corticosteroids are used to mask illness, especially lameness in horses. The detection of these drugs in equine body fluids requires accurate methods, particularly where misuse of corticosteroids is suspected. Gas chromatography/mass spectrometry (GC/MS) is well established as a reliable technique for the identification of drugs in biological fluids. Using GC/MS, we determined dexamethasone levels in horse urine and serum after intravenous application of a therapeutic dose. Dexamethasone was detectable, in serum for up to six hours, and in urine for up to 32 hours, after its administration. These findings indicate that serum measurements are unreliable for the detection of corticosteroid abuse, and demonstrate urine to be a more suitable body fluid for investigation. Nevertheless, it should be emphasized that, regardless of the technique employed, the clinical effects of dexamethasone last longer than 32 hours; thus, failure to detect dexamethasone does not disprove corticosteroid abuse.