Several methods are used to evaluate gastric motility in rodents, but they all have technical limitations. Recent technical developments enable a convenient method to evaluate gastric motility. The ¹³C-acetic acid breath test in rodents is a non-invasive and repeatable method that can be used without physical restraints. The present study aimed to validate the ¹³C-acetic acid breath test by measuring the effects of loperamide, morphine, mosapride, and itopride on gastric emptying in mice. Loperamide (1—10 mg/kg) and morphine (1.25—10 mg/kg) slowed gastric emptying and decreased the maximum concentration (C_max) and area under the curve (AUC_{90 min}) value in a dose-dependent manner. Mosapride (0.2—5 mg/kg) accelerated gastric emptying and increased C_max value. Mosapride (20 mg/kg) did not accelerate gastric emptying on the ¹³C-breath test. Itopride (30 mg/kg, per os) significantly accelerated gastric emptying compared with the vehicle group. In a comparison with the conventional phenol red test, there was a correlation between the C_max value of breath test and gastric emptying (%) of phenol red tests in treatment with loperamide or mosapride. These results indicate that the ¹³C-acetic acid breath test is an accurate, noninvasive, and simple method for monitoring gastric emptying in mice. This method is useful to assess the effect of drugs and gut function pharmacologically.