Sex identification through coelioscopy is a minimally invasive surgical technique used to determine the sex of chelonians by directly visualizing their internal reproductive organs. An adequate anaesthesiologic plan is essential to guarantee patient immobilization and proper analgesia during the entire surgical procedure. In this study, we evaluated the effects of a combination of dexmedetomidine (0.05 mg/kg), midazolam (1 mg/kg), ketamine (8 mg/kg), and morphine (1 mg/kg) (DMKM) randomly delivered intramuscularly (IM) or subcutaneously (SC) in twenty-one Aldabra giant tortoise (Aldabrachelys gigantea) into the right antebrachium for celioscopic sex identification. Heart rate (HR), respiratory rate (RR), and body temperature (BT) were measured, along with the skeletal muscle tone of the thoracic and pelvic limbs, neck retraction reflex, palpebral reflex, and jaw tone every 15 min. The anaesthesiologic plan was considered to be adequate at the loss of the thoracic and pelvic limb retraction reflexes. After a 45 min interval, if the anaesthetic plan was deemed insufficient for the celioscopic procedure, a 5 mg/kg dose of propofol was administered intravenously into the subcarapacial venous plexus. At the end of the procedure, atipamezole (0.5 mg/kg) and flumazenil (0.05 mg/kg) were administered intramuscularly into the left antebrachium as reversal agents. Both HR and RR decreased from baseline to both 15 and 30 min. Due to the persistence of thoracic and pelvic limb retraction reflexes 45 min after DMKM administration, 6/11 (55%) cases in the SC group required the additional administration of propofol, in contrast to only 1/10 (10%) cases in the IM group (p = 0.05). The recovery times were comparable between the successfully induced animals in the IM and SC groups. In this study, the intramuscular administration of a DMKM combination quickly produced chemical restraint, suitable for celioscopic sex determination.