Ultrasonic back-extraction of Triton X-100 reverse micelles by a water/chloroform binary system and gas chromatography with flame ionization detection (GC-FID) was developed for extraction and determination of β-sitosterol and cholesterol in soybean and sunflower oil samples. After the homogenization of the oil samples with Triton X-100, an aliquot of 200 μL of methanol was added to the samples to form two phases. The clear Triton X-100 extract obtained by centrifugation was treated with a mixture of water (1000 μL) and chloroform (300 μL) for back-extraction of the analytes into the chloroform phase by ultrasonication. After centrifugation, the sedimented chloroform layer was withdrawn easily by a microsyringe and directly injected into the GC-FID system. The influence of several important parameters on the extraction efficiencies of the analytes was evaluated. Under optimized experimental conditions, the calibration graphs were linear in the range of 1.0-30.0 mg L(-1) with coefficient of determination more than 0.994 for both analytes. The method detection limit values were in the range of 0.2-0.7 mg L(-1). The lower limit of quantification values were in the range of 0.7-2.4 mg L(-1). Intra-day relative standard deviations were in the range of 1.0-2.7%. This procedure was successfully applied with satisfactory results to the determination of β-sitosterol and cholesterol in spiked oil samples. The relative mean recoveries of oil samples ranged from 93.6% to 105.0%.