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Not AvailableBackground: Mycotoxins such as aflatoxins (AFs) and ochratoxin A (OTA) can pose severe health hazards because of their toxicity. Given a wide range of food matrices susceptible to fungal infections and possible cooccurrence of mycotoxins at different concentrations, validated multimycotoxin and multimatrix methods are strongly warranted. Objective: The aim of this research was to develop a simple and fast ultra-high performance LC (UHPLC) fluorescence detection (FLD)–based method to simultaneously determine AFs (B1, G1, B2, and G2) and OTA and, furthermore, to carry out single-laboratory validation in a range of cereals and processed product matrices. Methods: The sample preparation involved homogenization and extraction with methanol–water (80 + 20). For cleanup, an aliquot (3 mL) was diluted with phosphate-buffered saline, loaded on an immunoaffinity column (AFLAOCHRA PREP®), and eluted with methanol (1 mL). The cleaned extract was diluted with 0.2% acetic acid (at a 1:1 ratio) before injection into an ultra-high performance liquid chromatograph. To perform simultaneous analysis of AFs and OTA, the FLD program was developed by switching the excitation wavelength in a single chromatographic run. Results: The method provided LOQs of 0.25 and 1 ng/g for AFs and OTA, respectively, without involving any derivatization. In rice, the recoveries of AFs ranged from 84 to 106%, whereas OTA had a recovery above 72%, with the repeatability relative SDs