Reverse phase high speed liquid chromatographic methods are presented for the separation and detection of cephalosporins, penicillins, tetracyclines and other miscellaneous antibiotics. The reverse phase approach is superior to ion-exchange liquid chromatography and spectrophotometric, chemical and microbiological procedures currently in use. In addition to being simple and easy to control, the technique is rapid, convenient and precise and provides the basis for the direct analysis of pure compounds, stability samples, complex mixtures and dosage forms of all types. Preparative chromatography has been used in our laboratory for the separation and isolation of up to 500 mg of antibiotics. Using this approach, we have separated and isolated small impurities as well as pure feference compounds. The methodology reported here can be extensively applied to the separation, quantitation and isolation of both naturally occurring and synthetically produced antibiotics in a variety of media including physiological fluids.