This study was aimed at developing a single RP-HPLC method for determination of Natamycin in 20 different cheese samples purchased from local Turkish supermarkets. Chromatographic separation was achieved on a X-Terra RP18 column (250 mm x 4.6 mm x 5 µm) with a mobile phase of acetonitrile: water (30:70 v/v/v), at 0.8 mL/min flow rate with DAD detection at 305 nm. In twenty cheese samples, Natamycin was analyzed by using sample preparation method of ISO 9233-2, 2007 (IDF 140-2, 2007). The results of analysis have been fully validated statistically and recovery studies confirmed the accuracy of the proposed method. The precision (intra-day & inter-day) of method was found within limits (RSD < 2%). The sensitivity of the method was assessed by determining limit of detection and limit of quantification. Findings dealing with the presence of Natamycin in cheese samples are presented.