Abstract: A high performance liquid chromatography method was established for the determination of lutein and astaxanthin in Amphiprion frenatus. The sample was extracted by acetone and methyl tertiary butyl ether,and cleaned up by an LC-NH₂ column. The chromatographic separation was achieved on C₁₈column used amobile phase made up of methanol and water(98:2,v/v)by equal gradient elution. The detection wave length was set at 475 nm,and an external standard method was used to quantify astaxanthin and lutein. An excellent linear relationship of this method for astaxanthin and lutein were observed with values of 0.998 and 0.999. The average spike recovery rates of lutein and astaxanthin were 79.92%~90.32% and 78.51%~97.60% with relative standard deviations of 6.35%~7.07% and 10.79%~13.41%. This method was sensitive,repeatable and suitable for the determination of astaxanthin and lutein in small ornamental fish.