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A selective and sensitive method based on derivatisation with 2,4-dinitrophenylhydrazine (DNPH) and consecutive HPLC gradient separation is described for the determination of malondialdehyde (MDA) in urine. Preparation of urine samples involved a one-step derivatisation/extraction procedure. Separation was achieved using a Waters Symmetry™C₁₈ column (3.9×150 mm) and linear gradient of acetonitrile in water (from 30% to 70% in 30 min). The overall detection limit of the method was 56 nM of MDA in urine. The recovery of MDA was 94.3±8.6%. MDA in urine of healthy volunteers, measured using the method of standard additions, was 0.019±0.012 μmol/mmol creatinine. MDA in the same samples measured using the 2-thiobarbituric acid (TBA) assay was 0.181±0.063 μmol/mmol creatinine. We demonstrate that the commonly used TBA assay in conjunction with HPLC may overestimate the MDA …