Tetracyclines are a group of broad spectrum of antibiotics. Structure can be described as derivatives of polycyclic naphthacene carboxamide. Tetracyclines are hydrophilic and basic compounds due to the presence of hydroxyls and amino group. Mixture of tetracyclines and their impurities were separated based on weak reversed-phase and strong cation-exchange mechanisms on a Primesep 200 mixed-mode HPLC column. Elution of compounds was monitored by UV.