Twenty different macrolide and ketolide antibiotics were analyzed by reversed phase high performance liquid chromatography on an ODS-2 cartridge column. Each of these compounds was uniquely separated and purified by varying the flow rate. Retention times of the individual drugs were proportional to the flow rate of the mobile phase. Recovery of antimicrobial activity for most of the drugs was greater than 90% based on a microbiological assay of material recovered from the column. Retention times were related to structural differences between these antimicrobial agents.