This paper presents the development, optimization and validation of a new HPLC method used for the separation and determination of zearalenone, ZON, and its metabolites in biological samples of Leghorn broiler. ZON and its metabolites can be separated with good resolution in 11 min, using a Hypersil Gold C18 column, a mobile phase mixture of 50 mM aqueous ammonium acetate:acetonitrile:methanol, 45:8:47 (v/v/v), flow rate 1 mL/min and column temperature 40 degrees C. Based on the results obtained by this method applied on biological samples one can conclude that liver is the site for zearalenone localization and detoxification. Influence of zearalenone on the nutritional properties of broiler meat (weight variation, gross chemical composition, fatty acids profile of the meat) was studied, also. Results obtained during 4 days of treatment with ZON showed minimal or no effects of the dietary zearalenone on broiler meat nutritional quality.