Abstract For the first time, this study reports the content of tropane alkaloids (TAs) in soybean and soymeal samples sourced from 13 different countries, as well as in various cereals (including corn, millet, wheat, and sorghum) and a product (Super Cereals). The analysis was conducted using a fully validated solid-liquid extraction (SLE) with dilute-and-shoot (DnS) method along with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) triple quadrupole system. The samples were extracted using a solution of acetonitrile and water (60:40) with 1% formic acid. The limits of quantification (LOQ) were 0.10 µg kg−1 and 0.25 µg kg−1 for atropine and scopolamine, respectively. The extraction recovery was at least 80%, and the intra- and inter-day precision (relative standard deviation) were below 15% at 1, 5, and 50 µg kg−1 atropine and scopolamine spiked levels. The validation parameters showed that the method was fit for the purpose of accurate LC-MS/MS quantification of atropine and scopolamine. The SLE with DnS is high-throughput, enabling the extraction of TAs in 2.3 min per sample or at least 60 samples in 140 min. Whereas typical TA extraction methods require lengthy and costly clean-up and concentration steps, the DnS does not require these steps, sophisticated equipment, or an expert operator for execution, which makes it a simpler and straightforward procedure for TA analysis in different grain-based food matrices. Application of the LC-MS/MS method showed that one in 80 millet or corn, 18 in 269 soybeans, 20 in 62 soymeal and 20 in 42 Super Cereals samples had atropine and scopolamine above the maximum level of 1 μg kg−1 set by the EU Commission Regulation. TA contamination in these samples could either be due to the mixing with (visible) or exposure to (invisible) TA-producing plant material. The latter was proven through an exposure experiment, which showed that atropine and scopolamine from Datura stramonium seeds can be transferred to food grains (soybeans) invisibly with prolonged contact.