A proficiency test (PT) for the determination of the tropane alkaloids (TAs) atropine and scopolaminein buckwheat flour and maize flour was organised by the European Union Reference Laboratory for mycotoxins & plant toxins (EURLMP) between August and October 2020. This PT was carried out by Wageningen Food Safety Research (WFSR) in accordance with ISO/IEC 17043 (R013). The measurandlevels were targeted to provide insight on the measurement capabilities of the EU Member States’National Reference Laboratories (NRLs) at concentrations corresponding to the levels of TAs in processed cereal-based foods and baby foods for infants and young children, containing millet, sorghum, buckwheat or their derived products as regulated by Commission Regulation (EU) 2016/239. In addition to this food product, a maize sample containing a higher level of TAs was included in this PT since an amendment to the legislation is foreseen.The participants were asked to quantify atropine and scopolamine in two materials and to report the compounds individually as well as the sum value. The participants performance was assessed as z-score in both materials for the individual TAs (maximum score 4 out of 4) and for the sum of the two TAs in one sample (maximum score 2 out of 2).Thirty-eight participants, of which 29 NRLs for mycotoxins and/or plant toxins in food and feed (from 22 EU Member States plus Iceland and Norway) and 9 Official Laboratories (8 from 4 EU Member States and Switzerland) participated in the PT.Two materials, buckwheat flour (material A) and maize flour (material B), were prepared containing atropine and scopolamine. Levels were artificially increased by spiking with atropine and scopolamine standard solutions. Both materials were sufficiently homogeneous and stable during the PT. Each participant received one test sample of each material.For the identification and quantification of atropine and scopolamine 34 participants used liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS), one participant used LC single quadrupole MS, two participants used LC high resolution mass spectrometry (HRMS) and one participant provided no information. In this PT the robust mean was used as consensus value. The consensus value based on the participants’ results was used as the assigned value. The assigned values of atropine and scopolamine in material A were, respectively, 1.15 and 1.16 μg/kg and in material B, respectively, 15.3 and 52.7 μg/kg. Obtained interlaboratory reproducibility (RSDR) ranged from 14% to 26%. The RSDR were for all TAs below the target standard deviation, except for scopolamine (26%) in material A. For the sum of TAs (atropine and scopolamine) the RSDR was 20% and 17% for material A and B, respectively. The proficiency of the participants was assessed through z-scores, calculated using the assigned values and a relative target standard deviation of 25%. All participants submitted results for atropine and scopolamine. One participant analysed only material A. For both materials (A and B) 87% of the results for atropine and scopolamine were rated with satisfactory z-scores (|z|≤ 2), 6% of the results fell into the questionable range with 2<|z|<3 and 7% of the results fell into the unsatisfactory range with |z|≥ 3. Twenty-six participants achieved optimal performance for both materials by detecting both TAs with the correct quantification and the absence of false negative results. In this PT, two false negatives were reported.