The aim of this study was to compare the sedative and physiological effects following either oral transmucosal (OTM) or intramuscular administration of dexmedetomidine–methadone combination in healthy dogs. Thirty dogs were randomly assigned to receive a dexmedetomidine–methadone combination either by the OTM (n = 15) or intramuscular (n = 15) route. Sedation was scored 10, 20, and 30 min after drugs administration. Heart rate (HR), non-invasive blood pressure (NIBP), respiratory rate (fR), and body rectal temperature were recorded before drugs administration and then every 10 min for 30 min. Propofol dose required for orotracheal intubation was recorded. Sedation scores increased over time within both groups with higher values in intramuscular group (p (p p p R was lower compared with the OTM group at all the observational time points (p p < 0.05). Compared to intramuscular dexmedetomidine–methadone, OTM combination produced lower but effective sedation in healthy dogs.