An HPLC method for the analysis of water-soluble annatto in food was established. Water-soluble annatto was extracted from food samples with 1 % ammonia-70 % ethanol and the extract was then purified with a CIS cartridge after evaporation of ethanol before being subjected to the HPLC analysis. The HPLC conditions were as follows: column, TOSOH TSK gel ODS-80Ts (5μm, 4.6×250 mm) ; column temperature, 40℃ ; mobile phase, acetonitrile-0.01 mol/L trifluoracetic acid (74 : 25) ; flow rate, 1.0 mL/min ; detection wavelength, 454 nm. The average recoveries of the water-soluble annatto were over 71 % from juice and jelly fortified at the concentrations of 1000μg/g. The coefficients of variation were less than 5.3 %. The present method is considered to be useful for the analysis of water-soluble annatto in foods.