Josamycin is a macrolide antibiotic effective against gram-positive bacteria. It is highly polar and does not retain well on reversed-phase columns. The Mixed-mode column Obelisc R was used to retain josamycin and its related impurities. Primesep D was also used to analyze josamycin impurities because Primesep D does not retain high-molecular weight plasma components. This method is compatible with LC/MS, prep chromatography and can be used for analysis of josamycin in biofluids.