Background: There are limited studies on the utilization of analgesics in testudines. Management of pain in reptiles is by use of analgesics generally used in other vertebrate species. Evidently, some analgesics considered to be generally effective in reptiles are not effective in certain reptile species. Objective: The purpose of this study was to examine the effect of amitriptyline hydrochloride on nociceptive behaviour in Speke's hinge-back tortoise. Methods: Twenty-four adult Speke-hinged tortoises weighing 500–700 g were used. The effects of amitriptyline hydrochloride on nociception were evaluated using the formalin, capsaicin and hot plate nociceptive tests. Amitriptyline was administered intracoelomically at doses of 0.5, 1.0 and 3.0 mg/kg. Results: The higher doses of amitriptyline hydrochloride caused an increase in nociceptive behaviour (time spent in hindlimb withdrawal) on the formalin and capsaicin nociceptive tests, suggesting a potentiating effect. However, the doses used had no significant change in nociceptive behaviour on withdrawal response in the hot plate test. Conclusions: The study showed that amitriptyline hydrochloride which is widely used in management of neuropathic pain potentiates nociceptive effects in the formalin and capsaicin nociceptive tests in the Speke's hinge-back tortoise. The hot plate test, which previously has not been reported in these animals, gave results not in line with the other tests and therefore more testing and validation of the test is required. Amitriptyline modulates chemical and thermal pain differently.