bstract Wheat is one of the world’s most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.