A high-performance liquid chromatographic (HPLC) analysis method for the novel anti-HIV drug FP-21399 in human plasma was developed. The method employed the combination of organic solvent extraction and solid phase extraction. Analysis of FP 21399 and two major metabolites was achieved within 18 min using a reverse phase Puresil Cl 8 analytical column (4.6×150 mm, 5 μm, Waters) with a mobile phase of water-acetonitrile containing 20 mM triethylamine acetate (apparent pH 7.0). Linear gradient of mobile phase was applied as water-acetonitrile from 78:22 (v/v) to 55:45 over 8 min, and held at this ratio for the next 4 min. An ultraviolet-visible detector was operated at 265 mn from 0 to 8 min and at 600 nm from 8 min and after. The retention time of FP-21399 was 8.8 min and a linear response was observed over the concentration range 0.01–100 μg ml −1 ( r=0.994). Lower limit of quantitation was found to be 0.01 μg ml −1. Intra- and inter-assay precision varied in the range of 0.2 to 8% and 1–12%, respectively. The bias ranged from −17–3% for all analyses. A series of clinical plasma specimens were successfully analyzed using this method. The strategies for the method optimization on HIPLC separation and extraction procedure are discussed as well.