A high-performance liquid chromatographic procedure for the measurement of fifteen β-lactam antibiotics in body fluids is described, with special reference to high-speed techniques. The procedure involves a unique sample preparation before analysis for all the following fifteen compounds: benzyl-penicillin, ampicillin, cloxacillin, ticarcillin, mezlocillin, azlocillin, piperacillin, cefotaxine and its desacetyl metabolite, cefsulofin, cefoperazone, cefmenoxime, ceftazidime, ceftriaxone and the monobactam aztreonam; thus all biological samples arriving at the laboratory can be treated in batch. Of these fifteen antibiotics, eleven can be chromatographed with the same type of mobile phase, which consists of a mixture of ammonium acetate and acetonitrile in various ratios. Three others need ion-pairing chromatography because of their polarity, and ticarcillin requires citric acid. High-speed high-performance liquid chromatography seems to be particularly suitable for the routine analysis of β-lactam antibiotics because columns equilibrate more rapidly, retention times are much shorter, detection limits are lower and the longer lifetime of columns reduces analysis costs.