Adult gopher tortoises (Gopherus polyphemus) are difficult to physically restrain, particularly for examination of the upper respiratory tract and oral cavity—areas important for biological sample collection during disease surveillance studies. Collection of nasal lavage is required for determining the status of Mycoplasma infection in tortoises. Anesthesia is often necessary to provide full relaxation in order to gain access to the head and mouth for nasal lavage. Therefore, deep sedation or general anesthesia are required both for the welfare of the tortoise and to maximize the usefulness of biological samples collected. The objective of this study was to assess the utility and safety of a novel field-anesthetic combination of dexmedetomidine–ketamine–morphine (75 mc/kg; 8 mg/kg; 1 mg/kg, respectively), followed by reversal with atipamezole (0.02 mg/kg). Between May and October 2009 at the Joseph W. Jones Ecological Research Center in Baker County, Georgia, USA, 128 tortoises were captured and anesthetized with this protocol. Average time to first effects was 9.7 minutes, to induction was 21.6 minutes, to total recovery after administration of reversal was 92.7 minutes, and the mean time from administration of anesthesia through complete recovery for adult tortoises was 129 minutes but ranged from 40 minutes to 300 minutes. Clinical signs of upper respiratory tract disease significantly reduced the induction time such that tortoises with clinical signs lost consciousness faster than healthy tortoises. Study month significantly influenced the time to induction and time to recovery, such that as the study progressed, both parameters decreased. This anesthetic protocol proved to be an effective, reliable, repeatable, and safe method to collect quality biological samples from gopher tortoises. © 2013 The Wildlife Society.