A liquid chromatographic procedure is described for rapid and accurate quantitation of vancomycin in serum. Vancomycin is extracted from serum by using a CM-Sephadex® column and is measured by reversed-phase chromatography with detection by UV absorption. Ristocetin is used as an internal standard. Concentration and instrument response are linearly related in the range of therapeutically useful concentrations. Results correlate well with those obtained by bioassay. The between-day coefficient of variation (n = 8) was less than 4%. Recovery of vancomycin from serum is nearly complete, and no substances that interfere with the procedure have been detected in clinical specimens.