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The metabolism of the masking agent tolcapone in the horse has been investigated. This substance was found to have undergone various chemical transformations that produced a large variety of phase I metabolites, as well as glucuronide and sulfate conjugation. Confirmation of the presence of tolcapone and the 3‐O‐methylated metabolite in the blood samples collected up to 240 minutes and in urine obtained up to 24 hours, was successfully conducted using both gas chromatography– and liquid chromatography–tandem mass spectrometry techniques. The 3‐O‐methyl tolcapone is the better marker to use in a screening method because, in comparison to tolcapone, we have found that this substance offers superior chromatographic performance that should potentially give a lower limit of detection.