The purpose of this study was to evaluate the efficacy of dexmedetomidine and ketamine and reversal with atipamezole administered intranasally to Trachemys scripta scripta. Eight healthy adult turtles received 0.2 and 10 mg/kg of dexmedetomidine and ketamine, respectively. Heart rate, respiratory rate, temperature, and sedation (on a scale of 0–4) were measured. Blood was collected 45 min post induction for drug plasma levels from both the subcarapacial and tail veins, followed by intranasal atipamezole administration (0.5 mg/kg). The most frequent sedation scores (2 and 3) provided a level of sedation deep enough to perform a thorough physical exam and minor clinical procedures. All of the turtles became active in 18.9 ± 7 min after atipamezole administration. No adverse effects were observed and all measured cardiopulmonary parameters were within normal limits. Plasma levels of both dexmedetomidine and ketamine indicated adequate absorption and distribution with no difference in the levels obtained from either the subcarapacial or the tail venipuncture sites. A combination of dexmedetomidine–ketamine can be used intranasally to induce moderate to heavy sedation for physical examination, venipuncture, and other minor procedures in the yellow-bellied slider without adverse effects. In addition, the intranasal administration of atipamezole appears efficacious for reversal of dexmedetomidine. Intranasal administration of anesthetic agents holds promise for sedating and anesthetizing chelonians, which are often difficult to thoroughly examine and restrain for biological sample collection, both in the clinic and in the field.