The three-drug anesthetic mixture (medetomidine, midazolam and butorphanol), developed as an injectable anesthetic for laboratory animals, has been verified from various perspectives and applied to mice and other laboratory animals. However, the effects of its storage conditions and periods on its efficacy have not yet been studied. This study investigated the mixture's efficacy after storage under various conditions (room temperature, 4°C and -20°C) for 1 and 2 years. Mice in all groups were induced into a stable anesthetic state for at least 15 min after administration. The mice recovered from the anesthetized state 35 min after administration of the antagonist. These findings demonstrate the mixture's stability under different storage conditions and durations, potentially improving laboratory mouse welfare.