An electron-capture gas chromatographic procedure was developed for the simultaneous analysis of the enantiomers of fluoxetine and norfluoxetine. The assay involves basic extraction of these enantiomers from the biological samples, followed by their conversion to diastereoisomers using the chiral derivatizing reagent (S)-(-)-N-trifluoroacetylprolyl chloride. The method was utilized to detect and measure the quantity of these enantiomers in plasma and urine of patients and in liver and brain tissue of rats treated with (R,S)-fluoxetine.