A sensitive and selective analytical method for the quantitative determination of fumonisins B1 (FB1 ) and B2 (FB2 ) in maize-based foods for direct human consumption is described. The method, based on high-performance liquid chromatography and fluorescence detection, presents a rapid and automated online post-column derivatization, performed with o -phthalaldehyde and N ,N -dimethyl-2-mercaptoethylamine (Thiofluor™). A complete separation of fumonisins is achieved in less than 13 min by using a C18 column and a gradient elution. Fumonisins are extracted from the sample with a mixture of water, acetonitrile, and methanol. The filtered extract is purified by immunoaffinity column and FB1 and FB2 are eluted with methanol. The method has been successfully validated, and performances comply with �criteria of the Regulation EC No 401/2006.