Abstract: OBJECTIVE To establish an HPLC method for the study of tissue distribution of homoharringtonine in rats. METHODS The biological samples were extracted with chloroform in alkalinity condition. The Kromasil C18 column (4.6 mm×150 mm, 5 mm) was used as analytical column with a mobile phase consisted of acetonitrile-10 mmol·L^-1 phosphate buffer solution (0.2% triethylamine, pH 2.5) (25∶75) at a flow rate of 1.0 mL·min^-1 with fluorescence detector set at λex 280 nm and λem 320 nm. RESULTS The recovery of the method was 91.3%-102.4%, the LOD was 0.5 ng·mL^-1. In main effect tissues, the homoharringtonine concentration was followed as in order C_bm>C_coeur, and that was C_liver>C_lung>C_kidney in eliminated tissues. CONCLUSION The method has a good selectivity and reproducibility and can be used for the investigation of homoharringtonine in biosamples.