OBJECTIVE：To establish th e method for the determination of related substances in fidaxomicin raw material. METHODS：The detection ability of NP-HPLC-UV ，RP-HPLC-ELSD and RP-HPLC-UV systems for the related substances in fidamycin raw material was investigated and the best chromatographic system was selected . The HPLC detection method for the related substances was established. The detection was performed on Agilent Eclipse XDB C 18 column with mobile phase A consisted of 0.2％ triethylamine buffer solution （pH 3.8）-acetonitrile（55∶45，V/V），mobile phase B consisted of 0.2％ triethylamine buffer solution（pH 3.8）-acetonitrile（20∶80，V/V）at the flow rate of 1.0 mL/min（gradient elution ）；the detection wavelength was set at 230 nm，and column temperature was 35 ℃；the sample size was 10 µL. Calculation of the content of related substances was principal component self-control method without correction factor. RESULTS ：The impurities C and F could not be separated effectively in NP-HPLC-UV system. In RP-HPLC-ELSD system ，only impurities C ，D，E and F could be detected. In RP-HPLC-UV system ，11 impurities could be detected. In the study of methodology ，the linear ranges were 0.5-20.0 μg/mL for fidaxomicin（R2＝0.999 9）；the LOD was 0.05 ng，LOQ was 0.15 ng；RSDs of reproducibility and intermediate precision tests were less than 2.0％（n＝6）；average recovery was 98.4％（RSD＝3.6％，n＝9）. The sum of impurities in 3 batches of raw materials were 0.53％，0.51％，0.51％，respectively. CONCLUSIONS ：The effect of detecting impurities by RP-HPLC-UV are the best. Established method is specific and sensitive ，and can be used for the determination of related substance in fidaxomicin raw material.