The toxicity of some antibiotics used today necessitates rapid, specific and accurate determinations of antibiotic concentrations in biological fluids. Microbiological assays do not fulfill these needs and new procedures have recently been developed, for instance, fluorimetric assays, radioimmunoassays and radioenzymatic assays. However, these techniques have not been generally applicable to various antimicrobial agents. - The technique for high-pressure liquid chromatography (HPLC) has recently been applied to determination of antibiotics in biological fluids. The methods involve extraction of drug from the biological samples, separation by HPLC and detection by ultraviolet spectrophotometry of fluorimetry. Results obtained with tetracycline, amphotericin B, and cephalothin show that this procedure meets the demands for rapid, specific and accurate monitoring of antibiotic concentrations in body fluids. Because of its versatility, the HPLC technique seems to be applicable to the determination of a variety of antibiotics and other drugs in clinical and experimental medicine.