We wished to improve the efficacy and safety of ketamine/xylazine anaesthesia in C57Bl/6JRj mice and achieve a surgical anaesthesia lasting 20-30 minutes, aiming at fewer anaesthesia related deaths and using the subcutaneous injection route to inflict less stress and pain on the mice. This was achieved by adding midazolam to the ketamine/xylazine anaesthesia, at dosages of 193.55 mg/kg ketamine, 4.3 mg/kg xylazine and 3.76 mg/kg midazolam. This study demonstrated that subcutaneous administration of ketamine and xylazine in combination with midazolam resulted in surgical anaesthesia for at least 25 minutes. We also confirmed that oxygen supply is necessary to maintain physiological levels of peripheral blood oxygenation in order to avoid hypoxia during ketamine/xylazine anaesthesia.