This paper describes the development of a sensitive high performance liquid chromatography (HPLC) method for quantification of rosiglitazone in sheep plasma and amniotic fluid. Samples were prepared by liquid-liquid extraction using tert-butyl methyl ether, and rosiglitazone was quantitated by HPLC using a C18 column and fluorescence detector with an excitation wavelength of 247. nm and emission wavelength of 367. nm. The mobile phase consisted of ammonium acetate (10. mM, pH 5.2) and acetonitrile (56.5:43.5, v/v) with a flow rate of 1. ml/min. Ketoconazole was used as the internal standard (IS).The plasma calibration curve was linear over the range of 2.5-250ng/ml (mean r2=0.9940±0.0024; n=6) with accuracy of 99.4-102.8% over the calibration range. The intra-day and inter-day coefficient of variation (%CV, percent coefficient of variation) were in the range of 0.01-8.68% in sheep plasma. Similar performance was achieved for amniotic fluid. The described method was successfully applied to quantitate rosiglitazone concentrations in the pregnant ewe and her fetus.