In the present paper, we develop a methodology for antimony speciation in occupationally exposed human urine samples by high-performance liquid chromatography with hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS). The methodology was applied to the determination of Sb(V), Sb(III) and (CH3)3SbCl2 (TMSb(V)). Retention time of Sb(V), Sb(III) and TMSb(V) species were 0.88, 2.00 and 3.61 and the detection limits were 0.18, 0.19 and 0.12μg L-1, for 100μL loop injection respectively which is considered useful for elevated/occupationally exposed urine samples. Studies on the stability of antimony species in urine samples on the function of the elapsed time of preservation (4°C) and storage (-70°C) were performed. Results revealed that antimony species are highly unstable at -70°C, probably due to co-precipitation reaction. In this kind of matrix transformation during preservation time may occur, such as oxidation of Sb(III) to Sb(V) and transformation into species that do not elute from the column. EDTA shows that it is able to stabilize Sb(III) for more than one week of preservation time at 4°C avoiding co-precipitation during storage at -70°C. Finally the methodology was applied to occupationally exposed human urine samples. 25% of specimens present antimony levels (Sb(V)) of more than 5μg L-1.