A modified method for the determination of natamycin in natural cheese by high performance liquid chromatography (HPLC) was developed. A 10g sample was taken from the surface and from the inner part of the cheese. Each sample was homogenized with a stomacher, and natamycin in the sample was extracted with 100ml of mixed solvent of acetonitrile and phosphate buffer (28:72). The acetonitrile-phosphate buffer extracts were subjected to quantitation by reversed-phase HPLC with a 28: 72 mixture of acetonitrile and phosphate buffer (pH 6.8, 0.02M) as the mobile phase, and the absorbance was recorded at 305nm. The minimum detectable natamycin concentration was 0.2μg/g cheese and recovery of natamycin was determined to be more than 97.5% when 8μg/g natamycin was spiked in natamycin-free cheese.