Dexamethasone is a potent corticosteroid anti-inflammatory agent, and in most jurisdictions it is not legal to administer to any horse on the day of a race. This study was conducted to determine whether topical combination of dexamethasone and dimethyl sulfoxide (DMSO) solutions would result in detectable dexamethasone in the blood or urine in horses. Five different concentrations of dexamethasone/DMSO were used to replicate the combinations used on race horses. Serum cortisol concentrations were determined to detect an alteration in the hypothalamus-pituitary-adrenal axis. Dexamethasone/DMSO mixtures were applied topically to the distal limbs in five Standardbred mares. Blood and urine samples were collected at 0, 8, 24, and 32 hours. Samples were originally screened by a commercially available enzyme-linked immunosorbent assay (ELISA) test for dexamethasone. Any samples that were deemed suspect were then analyzed by liquid chromatography/mass spectrometry (LC/MS). Blood cortisol was assayed using a solid-phase chemiluminescence enzyme immunoassay. There was no detectable dexamethasone in either the urine or serum of any horse at any time point. The serum cortisol concentrations were within our laboratory's normal range at all time points. It appears doubtful that detectable blood and urine concentrations of dexamethasone are attributable to absorption from a topical application through intact skin.