In this paper, a procedure for the determination of aflatoxin B1, B2, G1 and G2 in five different animal feedstuffs, intended to feed different mammalians and fowls, has been developed and validated. The method is based on a very simple solid–liquid extraction with acetonitrile and subsequent analysis by high performance liquid chromatography coupled with fluorescence detection with post-column photochemical derivatization. The study carried out to optimize the extraction step, showed that using acetonitrile as extraction solvent provided not only satisfactory recoveries, but also extracts clean enough to omit a further clean up step. The method has been fully validated on five different matrices, and limits of quantification were below the allowed or recommended levels by European Union. Recovery studies were carried out at three different concentration levels, with values ranging from 81 to 105%; repeatability and intermediate precision showed relative standard deviations lower than 10% in all cases.