Abstract: OBJECTIVE To optimize the determination of metronidazole in human milk and establish a limit test method. METHODS The experiments scheme was scheduled by uniform design method to improve the extraction of metronidazole from human milk, and the metronidazole was determined by HPLC method. RESULTS For extracting metronidazole from milk, the NaOH should not be added and the super sonic treatment could be removed. In the results of the validation experiments of the limit test method, there was no corresponding peak in the chromatograph of the control comparing with the chromatograph of the sample and the standard, the limit of detection was lower than 0.02 μg·L^-1, and the RSD of the test results of the same sample in different conditions was 2.2%. CONCLUSION Uniform design method is effective and rapid to improve the parameters of the test method. The limit test method of metronidazole in human milk established is sensitive, simple, rapid and reproducible.