An efficient method is described for the simultaneous determination of hair polyamines, such as 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine, by gas chromatography-mass spectrometry (GC-MS) with the selected ion-monitoring (SIM) mode. The method is based on the extractive two-phase ethoxycarbonyl (EOC) reaction of amino functions in aqueous solutions combined with subsequent pentafluoropropionyl (PFP) derivatization of the remaining active hydrogen atoms for the direct analysis by GC-SIM-MS. The detection limits for SIM of the polyamines as N-EOC-N-PFP derivatives ranged from 1 to 10 ng/g hair, while their recovery rates varied in the range of 76.42-93.38%. This method demonstrated a good overall accuracy (% bias) and precision (% C.V.) as 3.32-11.05% and 5.88-14.71%, respectively. When applied to 0.6 M HCl extracts of hair samples from 11 healthy men and 19 healthy women, all five polyamines were positively detected at the concentrations of 8.82-871.87 ng/g. Both in median and mean concentrations, the most abundant hair polyamine was spermidine, followed by spermine, putrescine, 1,3-diaminopropane and cadaverine in the male group, while the order of 1,3-diaminopropane and cadaverine was reversed in the female group. The levels of polyamines, except for cadaverine, in hair specimens studied were found to be higher in men than in women.