A generally used protocol for ketamine/xylazine anesthesia in mice consists of 100mg/kg ketamine and 10 mg/kg xylazine for intraperitoneal (IP) injection. It is our experience that this protocol does not provide satisfactory results as many of the mice needs to be supplemented to achieve surgical anesthesia. Moreover, IP injections are associated with pain, peritoneal irritation and the risk of perforation of abdominal organs. Subcutaneous (SC) injection removes the risk of peritoneal irritation and perforation of abdominal organs, as well as lowering levels of pain and stress caused by fixation. This study presents the results of a refined method for ketamine anesthesia in mice. Five male C57Bl/6Rj mice were injected IP with ketamine and xylazine (KX-IP), five males were injected SC (KX-SC) and five males were injected SC with KX and midazolam (KXM-SC). To examine the effect of oxygen on peripheral oxygen saturation and heart rate, a follow-up study was performed, including SC dosing only, using both males and females. The results showed that all animals (five) injected with KXM-SC, two out of five with KX-SC and none with KX-IP achieved surgical anaesthesia within 10 minutes. In the follow-up study, six males and five females (out of eight) with KX-SC and all animals (eight) with KXM-SC achieved surgical anaesthesia within 10 minutes. Moreover, providing oxygen had a positive effect on peripheral oxygen saturation and heart rate. It was concluded that refined anaesthesia with KXM-SC is possible, but that oxygen must be supplied for optimal results.