Ergosta-4,6,8(14),22-tetraen-3-one (ergone) isolated from <i>Polyporus umbellatus</i> possesses a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic, and immunosuppressive effect. The interaction of cerium ions (Ce<sup>3+</sup>) with ergone was studied by fluorescence and absorption spectroscopy. Spectra data revealed that Ce<sup>3+</sup> ions exhibited emission maxima around 350 nm when the excitation wavelength was fixed at 255 or 290 nm, and the fluorescence of Ce<sup>3+</sup> ions was quenched by the addition of ergone, indicating that a Ce<sup>3+</sup>-ergone complex was formed. According to the modified Benesi-Hildebrand equation, the binding constant of interaction of Ce<sup>3+</sup> ions with ergone was obtained at room temperature. Based on this, a sensitive spectrofluorometric method using Ce<sup>3+</sup> ions as a probe was applied for the identification and quantification of ergone in rat plasma, feces, and urine. The linear ranges of the calibration curves were 1.31 to 4.50 µM for plasma, 1.12-9.87 µM for feces, and 1.28-3.42 µM for urine, and the ergone recoveries were found to be 97.1 ± 0.9%, 98.2 ± 0.7% and 96.5 ± 1.4% for plasma, feces, and urine, respectively. The intraday and inter-day relative standard deviations were less than 9.7%. The proposed spectrofluorometric method is simple and rapid for the quantitative determination of ergone in rat plasma, feces, and urine, and it is affordable for most laboratories because it has few requirements and uses low cost, easy to operate equipment.

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