A high-performance liquid chromatography method with pre-column derivatization for separation and quantification of allantoin in blood samples is described. Plasma after deproteinisation with trichloroacetic acid was used for the derivatization procedure. The procedure was based on allantoin conversion to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. Allantoin derivatives (syn- and anti-isomers) were separated on a reversed phase column (Nova-Pak C₁₈, 4 µm) by gradient elution, and then monitored at 360 nm. All components were completely resolved in about 46 min. The average recovery of allantoin added to plasma samples was 101.3 + 8.7% (n = 52). With UV detector the smallest allantoin concentration that gave reproducible integrations was 0.93 µmol/l. The within-assay coefficient of variation CV for derivatization and injection was 2.7 ± 1.2%, while CV for repeated injections was 0.68 ± 0.33%. This HPLC method can also be used for determination of allantoin in urine.