An isocratic high-performance liquid chromatographic method that uses aluminum oxide as the stationary phase is developed for the quantitation of creatinine in human serum. Sample pretreatment includes ultrafiltration, and ultraviolet detection is performed at 240 nm. The within-day precision, expressed as a mean coefficient of variation (CV), is 0.8%; the total method CV is 1.2%. The inaccuracy of the method is +1.5, -0.3, and +0.5%, respectively, as determined with the National Institute of Standards and Technology standard reference materials 909, 909 a1, and 909 a2. The detection limit of the method is 1.6 pmol (180 pg). The absence of interferences is confirmed by chromatographically analyzing patient serums again after enzymatic breakdown of creatinine.