Abstract The good correlation between exposure to n-hexane and 2,5-hexanedione urinary excretion confers on this diketone an important toxicological meaning. This paper proposes a reversed-phase HPLC method which includes, after acid hydrolysis, a derivatization step of 2,5-hexanedione with 2,4-dinitrophenylhydrazine at 70°C for 20 min. The reaction conditions, such as temperature, reagent concentration and time, are optimized so as to allow the condensation of a single carbonyl group. A linear response was obtained in the 0.19-20.0 mg/l range with a detection limit of 0.03 mg/l, corresponding to a signal-to-noise ratio of 3. A phosphate buffer (pH 3.3)-acetonitrile mixture (50:50) as the eluent and UV detection at 334 nm were used.