Abstract: A method was developed to rapidly analyze complex multicomponent in a novel antibiotic bitespiramycin. A Kromasil C 18 column (200×4.6 mm, 5 μm particle size) was used. The mobile phase consists of acetonitrile -10 mmol/L ammonium acetate ( V (acetonitrile)∶ V (ammonium acetate)=60∶45). An electrospray ionization source was used for mass analysis and detection. The full scan and two stage scans were performed in the positive ion detection mode. The results showed that ten spiramycin derivatives were found in bitespiramycin. Their chemical structures were deduced and identified by the reference substances of them, respectively. It indicated that high performance liquid chromatography mass spectrometry was successfully applied to profile components in a novel antibiotic bitespiramycin and should be intended as a simple and convenient way for the high throughput analysis of screening novel macrolide antibiotics.