Atrasentan is an endothelin antagonist selective for the ET A receptor in development at Abbott Laboratories for the treatment of cardiovascular disease and cell proliferation disorders. A simple and sensitive chromatographic method for the determination of atrasentan in human plasma has been developed and validated. The analytical method involves acidification of the plasma samples with 0.3 N HCl prior to extraction with 1:1 (v:v) hexane/tert-butylmethylether. The organic extract was evaporated to dryness, reconstituted with 20:80 (v:v) acetonitrile/0.05 M K 2 HPO 4 and washed with 75:25 (v:v) hexane/tert-butylmethylether. The organic layer was discarded and the aqueous layer was injected into the HPLC. Atrasentan and internal standard (ABT-790) were separated from interference using a 250 Γ 4.6 mm, 5 mm, 120 A Λ, Phenomenex Spherisorb C 8 analytical column with a 50 Γ 4.6 mm, Alltech Absorbosphere 5 mm CN guard cartridge using a mobile phase consisting of 25:15:5:55 (v:v:v:v) acetonitrile/isopropanol/ methanol/0.05 M K 2 HPO 4 , pH 7.0, at a flow rate of 1.0 mL/min. Fluorescence detection was achieved using l ex 278 nm and l em 322 nm. For a 1.0 mL plasma sample volume, the limit of quantitation was approximately 200 pg/mL. The method was linear from 0.2 to 1300 ng/mL (r 2 = 0.9986). Inter-and intra-day assay RSD (n = 6) were less than 10%. Mean accuracy determinations showed the quality control samples to range between 94 and 99% of the theoretical concentration.