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 significantly by the substitution of high efficiency, small particle (-10 pm) 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.In a previous paper" we described conditions for the determination of cephalosporins, penicillins, tetracyclines and other miscellaneous antibiotics by reverse phase high speed liquid chromatography.A second paper 2) dealt with a specific application of this technique to the determination of cephalexin and cephradine in various pharmaceutical formulations and in physiological fluids.