Several methods are currently available for quantitation of antimicrobial drugs in serum and body fluids, e.g., microbiological, radioenzymatic, and radioimmune assays. Recently, high-pressure liquid chromatography (HPLC) has been implemented for quantitation of a variety of drugs, including antimicrobial drugs. The general principles of chromatography, particularly in reference to HPLC, are reviewed. The methods and procedures for extraction, separation, detection, and quantitation of antimicrobial drugs by HPLC are discussed. Current HPLC techniques for measuring antimicrobial drugs in biological fluids are also summarized.