Objectively recognizing postoperative pain in mice is challenging, making it difficult to determine an appropriate postoperativeanalgesic regimen. Adult male mice produce ultrasonic vocalizations after exposure to adult female urine (FiUSV). To determine if FiUSV can be used as a indicator of postoperative pain, FiUSV produced by male C57BL/6J mice were assessed for 5 d before and after vasectomy or sham surgery with or without sustained-release buprenorphine. Postoperative pain was assessed by monitoring vocalization using an ultrasonic microphone and by evaluating orbital tightness, posture, and piloerection at postoperative time points. Before vasectomy or sham surgery, 25 of 38 male mice produced FiUSV on 4 of 5 d(143 ± 93 FiUSV). Vasectomized mice without postoperative analgesia produced significantly fewer FiUSV (59 ± 26 FiUSV) compared with baseline (212 ± 102 FiUSV) at 4 h postoperatively, but returned to baseline by 28 h. Vasectomized mice treated with buprenorphine and sham-surgery mice had no change in FiUSV from baseline at any time point after surgery. Activity was decreased compared with baseline in vasectomized mice, regardless of receiving postoperative analgesia or not, but only at the 4-h time point. There were no differences in behavior scores between vasectomized mice and sham-surgery mice at any time point. These results show that FiUSV can be used to detect postoperative pain in male C57BL/6J mice after vasectomy.