In this research a new method was developed and optimized for the determination of Sb(V) and Sb(III) in human erythrocytes fractions (plasma and cytoplasm) by high performance liquid chromatography with hydride generation atomic fluorescence spectrometry. The method considers the first step of samples cleaning by protein precipitation by salting out followed by C18 solid phase extraction, EDTA elution, and finally a chromatographic separation by using anion exchange PRPX-100 (100 mm×4.1 mm) and EDTA 20 mmol L-1 as mobile phase. The method was optimized by experimental design with a recovery of 90% for Sb(V) and 55-75% for Sb(III) approximately. The analytical method was applied to study the distribution of Sb(V) and Sb(III) in human erythrocytes considering temperature and time of incubations and with special attention about the influence of the anticoagulant. Results showed that both Sb(V) and Sb(III) are capable to enter the red blood cell in a proportion of approximately 40-60%. On the other hand, both species are then excreted from the interior of the cell, where the percentage considerably decreased from approximately 60 to less than 30% within the cell. An increase in the culture temperature increases the capacity of Sb(V) and Sb(III) to penetrate the membrane barrier and reach the cytoplasm. In order to preserve the original distribution of Sb in blood, heparin seems to be the best anticoagulant for sample preservation.