Plasma concentrations of dexmedetomidine (D = 0.1 mg/kg), midazolam (M = 2 mg/kg), and butorphanol (B = 0.4 mg/kg) were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) after their simultaneous (DMB) transnasal (TN) administration to healthy rabbits. Time-dependent changes in sedation and antinociception were evaluated by measuring a sedation score based on rabbit's posture, loss of the righting, palpebral and pedal withdrawal reflexes and by instrumental monitoring of rectal temperature, heart rate, arterial blood pressure, pulse-oximetry, and capnometry. The peak plasma concentration (Cmax ) of each drug was reached within 5 min (Tmax ) from DMB-TN administration along with deep sedation and analgesia. Such effects subsided after 45 min into a moderate sedation and analgesia lasting for additional 15 min. All rabbits awakened spontaneously and uneventfully 90 min after DMB-TN administration. During the anesthetic procedure, arterial blood pressure markedly decreased and respiratory depression ensued requiring oxygen supplementation. The results of this study show that all three molecules of the DMB combination were absorbed through the TN route, inducing deep sedation and analgesia suitable for minor surgical procedures. Such combination should be used with caution in rabbits bearing cardiovascular or respiratory diseases because of its ability to induce hypotension and respiratory depression.