The origin of caffeine detections in equine serum and urine after theophylline administrations was examined. Three different preparations containing theophylline were administered to standardbred mares. Both blood and urine samples were collected. Caffeine was detected and quantified in theophylline administration samples by high performance liquid chromatography (HPLC) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). Further in vitro analysis showed that caffeine metabolites were not detected when caffeine, or caffeine-containing products, were added to urine. Data derived from HPLC-UV and LC–MS–MS analysis of dosages of theophylline and caffeine are used to propose the establishment of a threshold limit to control and discern between metabolic and administered caffeine concentrations. A serum caffeine concentration of 250 ng/mL and a urine caffeine concentration of 1000 ng/mL are suggested. Based on the data supplied, these threshold concentrations could effectively control orally administered caffeine in racehorses, up to the dosage used in this work, up to 72 h before sampling time.