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The structural and functional diversity of plant metabolites is largely created via chemical modification of a basic backbone. However, metabolite modifications in plants have still not been thoroughly investigated by metabolomics approaches. In this study, a widely targeted metabolite modificomics (WTMM) strategy was developed based on ultra‐high performance liquid chromatography‐quadrupole‐linear ion trap (UHPLC‐Q‐Trap) and UHPLC‐Q‐Exactive‐Orbitrap (UHPLC‐QE‐Orbitrap), which greatly improved the detection sensitivity and the efficiency of identification of modified metabolites. A metabolite modificomics study was carried out using tomato as a model, and over 34,000 signals with MS2 information were obtained from approximately 232 neutral loss transitions. Unbiased metabolite profiling was also performed by utilizing high‐resolution mass spectrometry data to annotate a total of 2,118 metabolites with 125 modification types; of these, 165 modified metabolites were identified in this study. Next, the WTMM database was used to assess diseased tomato tissues and 29 biomarkers were analyzed. In summary, the WTMM strategy is not only capable of large‐scale detection and quantitative analysis of plant‐modified metabolites in plants, but also can be used for plant biomarker development. [ABSTRACT FROM AUTHOR]