An new and simple method to determine the antimony (Sb) in cells using Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS) was established for studying the mechanism of metal resistance in the protozoan parasite Leishmania. The detection limit was 0.08 microgram/L respectively. The average recoveries at different concentrations were 92.8%-103.6%. This method was simple, precise, reliable. The obtained results were compared with those determined by ICP-MS, and no obvious differences could be observed. This method has been successfully applied to the mechanism study of metal resistance in Leishmania.