Improved methods for the separation and quantitation of cephalosporins, penicillins, tetracyclines and several miscellaneous antibiotics by reverse phase high speed liquid chromatography are presented. The methods have been improved significant]y by the substitution of high efficiency, small particle (-10μm) reverse phase columns in place of the previously used medium efficiency, pellicular columns. The conditions and procedures described here illustrate that considerable improvements in separation and sensitivity of detection of antibiotics are achieved. Pure compounds, complex mixtures of antibiotics in a variety of dosage forms and fermentation broths are routinely analyzed by the described procedures.