1 2,5-Hexanedione (2,5-HD), the main metabolite of n-hexane was extracted from urine samples using cold fiber head space solid-phase microextraction (CF-HS-SPME) based on thermoelectric cooling and analyzed with gas chromatography equipped with a flame ionization detector (GC-FID). Important parameters for improved efficiency of fiber sorption including sample volume, extraction time and temperature were optimized using a central composite design. The optimum conditions were determined, under which the detection limit and the relative standard deviations were found. The method was shown to be rapid, sensitive and easier than conventional methods for quantitative analysis of 2,5-HD in urine samples.