Among the sixteen-membered ring macrolide antibiotics containing multicomponents, midecamycin acetate modified from midecamycin as shown broadspectrum of antimicrobacterial activity and its metabolites also showed certain degree of antibacterial activity although they gave higher MIC values than midecamycin acetate. Especially midecamycin acetate has no bitter taste, low toxicity for liver, kidney and gastro-intestinal organs. Our study an this paper was to develop a high performance liquid chromatographic method for assay of midecamycin acetate. So we attempted to determine the potencies of midecamycin acetate and its preparations by HPLC in comparison with microbiological assay. The analysis of midecamycin acetate and its preparations was achieved by HPLC method using a μ -Bondapak C_(18) column and acetonitrile - methanol - IPC B_6 as a mobile phase. The column effluent was monitored at 231nm by UV-spectrophotometer. The retention time of midecamycin acetate was less than 4 minutes. So this method was very simple and rapid. Therefore we obtained better result for assay of midecamycin acetate by HPLC method than that of microbiological method.