We present a candidate Reference Method for determining creatinine in serum, based on isocratic HPLC. The chromatographic column, with alkaline-treated aluminum oxide as stationary phase, is eluted with an equivolume mixture of methanol/acetonitrile containing 70 mL/L aqueous NaOH (10 mmol/L), and ultraviolet absorbance is detected at 240 nm. We investigated the ruggedness of the method and validated its performance with isotope dilution gas chromatography-mass spectrometry (ID GC-MS) for a set of 22 patients' sera and 10 commercially available lyophilized control materials. The mean deviation from ID GC-MS was +0.1% (range, -2.2% to +2.2%). The between-day CV, calculated from six independent measurements performed on three different days, was 0.9% (range 0.2% to 1.9%); the within-run CV was 0.8%. The total error of the method was < 3%. These performance characteristics make the method suitable for target-setting of quality-control materials and accuracy assessment of routine test kits. For the latter application, done with split-sample measurements of a panel of 83 patients' specimens, we used the Boehringer Mannheim enzymatic creatinine PAP test on the Hitachi 911, the Kodak Ektachem single-slide enzymatic creatinine test on the Ektachem 700, the Merck Jaffé kinetic assay on the Mega analyzer, and the Roche Jaffé kinetic test on the Cobas Mira.