When cultured with Aspergillus niger, asiaticoside, purified from Centella Asiatica (L.) Urban, was metabolized into four metabolites including one novel alkaloid. A simple and rapid SPE-HPLC method for simultaneous determination of asiaticoside and its four metabolites in culture of A. niger was developed. The chromatographic separation was achieved on a Dikma Diamonsi C18 ODS column (200×4.6 mm i.d.) by gradient elution with 0.1% phosphoric acid in water and 0.1% phosphoric acid in methanol as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 204 nm and the column temperature was kept at 28 °C. The retention times of asiaticoside, asiatic acid, M1, M2, and M3 were 11.3, 9.4 and 17.1, 19.3 and 7.9 min, respectively. The mean recoveries of five analysts were all over 99% Quantification limits were 0.02 ug/mL for all tested compounds. The method was sucessively applied to the quantification of the biotransformation of asiaticoside.© 2011 IEEE.