Trypsin is a key intestinal endopeptidase and the most widely used enzyme in proteomics workflows. However, the influence of protein modifications—commonly introduced during food processing—on intestinal digestibility and allergenic potential remains underexplored. Traditional methods for evaluating protease cleavage efficiency primarily consider the local amino acid sequence context, overlooking post-translational or process-induced modifications. In this study, a novel proteomic strategy was applied to assess trypsin cleavage efficiency at modified versus unmodified scissile sites within Ara h 1, a major peanut allergen. Among the 17 modified residues analyzed, 7 (41%) exhibited significant differences in cleavage efficiency (≥20%). Modifications such as methylation, methylation with carbamoylation, formylation, and dihydroxylation were associated with impaired digestion. Reduced cleavage at these sites may enhance the sensitizing potential of the resulting peptides. These findings support the development of machine learning algorithms and the mining of public proteomic datasets to systematically identify modification-driven digestion resistance or facilitation.