Abstract: A HPLC method for the determination of natamycin in food was studied and reported in this paper.Natamycin in sample was extracted with methanol-water (3+1) at pH 4.5,natamycin in the extract was then determined by HPLC detected at 305 nm using an UV detector.Qualitative analysis was based on retention time and quantitative determination based on calibration curve.Recovery was in range of 95.7%-102.0%.RSD′s were less than 3%.Detection limit of the method was 0.50 μg·g-1 for solid sample and 0.25 mg·L-1 for liquid sample.