QXOH‐LB, a fixed‐dose combination (35 mM QXOH and 10 mM levobupivacaine) has been shown to induce a long duration of local anesthesia in animal efficacy testing, which indicates potential for postoperative pain management. In this study, we evaluated the potential toxicity of QXOH‐LB in NIH mice under the Guidance on the repeated‐dose toxicity published by the China Food and Drug Administration. Mice (n = 30 per sex per group) were subcutaneously injected 5, 10, 20 mg/kg QXOH‐LB, 5, 10, 20 mg/kg QXOH, and 5 mg/kg levobupivacaine (LB) once a day for 14 days with sacrifice of main study animals; remaining mice (n = 10 per sex per group) were monitored for an additional 4‐week recovery period. Mice in the 10 and 20 mg/kg QXOH, and 20 mg/kg QXOH‐LB died, which was considered due to excessive respiratory inhibition. The doses of 10 mg/kg QXOH‐LB and 5 mg/kg QXOH were well tolerated without any clinical signs of toxicity. Therefore, the no‐observed‐adverse‐effect level (NOAEL) of QXOH‐LB and QXOH was considered to be 10 and 5 mg/kg/day, respectively. In the dose range from 5 to 20 mg/kg, the exposure of QXOH and LB in QXOH‐LB was equal to each agent used alone at the same dose in NIH mice. There was no gender difference on exposure and no evidence of accumulation.