A high-performance liquid chromatographic (HPLC) method utilizing ultraviolet absorbance coupled with radioisotove detection was developed for the precise and simultaneous determination of iloprost and misoprostol. This assay allows complete resolution of iloprost diastereoisomers and has a total run time of approximately twenty minutes. Samples were prepared for chromatographic analysis by extracting a mixture of tritiated drugs from rat plasma with acetonitrile. The resulting solutions were chromatographed on a reversed phase Zorbax Rx-C8 column using 0.02M potassium phosphate (pH 3.0), acetonitrile, and methanol (46:30:24, v/v) at a flow rate of 1.7 mL/min. 2-Naphthoic acid was employed as an internal standard. The correlation coefficient for varying concentrations of tritiated iloprost (12.7 Ci/mmol specific activity) from 2.18 ng/mL to 21.8 ng/mL was 0.995, and the correlation coefficient for concentrations of tritiated misoprostol (50 Ci/mmol specific activity) from 0.617 ng/mL to 6.17 ng/mL was 0.993. The high selectivity and sensitivity of this assay make it useful for the simultaneous quantitation of iloprost and misoprostol.