The paper describes the development of a simple and highly selective method for the determination of deoxynivalenol (DON) in food and feed samples. It combines sample cleanup with sol-gel immunoaffinity columns containing monoclonal anti-DON antibodies and quantification of DON by high-performance liquid chromatography with ultraviolet detection. The sol-gel immunoaffinity columns are as selective as commercial DON immunoaffinity columns but superior with regard to production costs, storage stability, and reusability. In applying the method for the analysis of maize, wheat, and spaghetti samples, it offers detection limits (LOD, S/N = 3) of 240, 200, and 207 ng/g, and recoveries of 83, 99, and 97%, respectively.