The application of supercritical fluid extraction with carbon dioxide and modified carbon dioxide for the determination of fluconazole from an animal feed was studied. A fractional factorial design approach was used to examine the significant experimental variables for quantitative extraction of fluconazole. Gas chromatography with either flame ionisation or mass selective detection was used for quantitation of the extracts. The results indicated that modifier (methanol) had the greatest effect on the recovery of fluconazole from the animal feed.