Background: Studies in human medicine have concluded that acetazolamide reduces pain associated with carbon dioxide insufflation during laparoscopic surgery. However, there are no published reports regarding the use of acetazolamide for this purpose in companion animals, despite the increasing popularity of laparoscopic techniques in veterinary medicine due to their advantages over open surgeries.Objectives: Thirty mixed-breed female dogs were included in the study and randomly assigned to one of three groups: OVE (median celiotomy ovariectomy; n = 10), OVEL (laparoscopic ovariectomy, n = 10) and OVELA (laparoscopic ovariectomy with acetazolamide preoperative administration; n = 10). Experienced surgeons performed all procedures, and the anaesthetic and analgesic protocols were identical for all animals.Acetazolamide was administered orally (at a dose of 25 mg/kg) 2 h prior to induction in the OVELA group. Postoperative pain was evaluated using serum cortisol, salivary cortisol, and the University of Melbourne Pain Scale (UMPS) Score.Results: Any statistical differences were observed in the UMPS scores when the OVELA group was compared to the OVEL group at 1 h after surgery (p = 0.515), 12 h (p = 0.375) and 24 h (p = 0.242). Animals undergoing open surgery (OVE group) had significantly higher pain scores at all times after surgery when compared with OVEL and OVELA groups. A high positive correlation (r = 0.792; p = 0.01) was found between serum and saliva cortisol concentrations. Mean saliva cortisol concentration was not significantly lower for the OVELA group compared to the other groups.Conclusions: This study found evidence that preoperative administration of acetazolamide may be beneficial in managing postoperative pain in dogs after laparoscopic surgeries. However, further research with a larger sample size is needed to confirm this and to determine if acetazolamide should be included in a multimodal postoperative analgesia protocol for laparoscopic ovariectomy in dogs.