We describe a sensitive high pressure liquid chromatographic (HPLC) procedure for the analysis of the new beta-lactam antibiotic moxalactam. Conditions are described for either measurement of total drug concentration or the concentration of the individual isomers. The proteins in a 1.0 ml plasma sample are denatured with isopropyl alcohol which is then extracted into a chloroform reagent, leaving the drug in the aqueous phase. An aliquot is then injected into a mu-bondapak phenyl column. A similar extraction procedure was employed for tissue homogenates. Linear regression analysis and comparison of the HPLC assay with the microbiological assay gave a correlation coefficient of 0.97. Analysis of tissue samples indicated that significant concentrations of moxalactam were obtained at the site of infection.