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Tropane alkaloids are a wide group of substances that comprises more than 200 compounds occurring especially in the Solanaceae family. The main aim of this study is the development of a method for the analysis of the principal tropane alkaloids as atropine, scopolamine, anisodamine, tropane, tropine, littorine, homatropine, apoatropine, aposcopolamine, scopoline, tropinone, physoperuvine, pseudotropine and cuscohygrine in cereals and related matrices. For that, a simple solid-liquid extraction was optimized and a liquid chromatographic method coupled to a single stage Exactive-Orbitrap was developed. The method was validated obtaining recoveries in the range of 60–109% (except for some compounds in soy), precision values (expressed as relative standard deviation) lower than 20% and detection and quantification limits equal to or lower than 2 and 3 μg/kg respectively. Finally, the method was applied to the analysis of different types of samples as buckwheat, linseed, soy and millet, obtaining positives for anisodamine, scopolamine, atropine, littorine and tropinone in a millet flour sample above the quantification limits, whereas atropine and scopolamine were detected in a buckwheat sample, below the quantification limit. Contaminated samples with Solanaceaes seeds ( Datura Stramonium and Brugmansia Arborea ) were also analysed, detecting concentrations up to 693 μg/kg (scopolamine) for contaminated samples with Brugmansia seeds and 1847 μg/kg (atropine) when samples were contaminated with Stramonium seeds. [ABSTRACT FROM AUTHOR]