Mass spectrometry-based peptidomics provides a comprehensive approach to delineate the global proteolytic landscape of diseases and to identify potential biomarkers. However, current data analysis methods struggle to distill the peptidomic signatures of diseases. In this study, we conducted a peptidomic analysis of plasma from individuals with β-thalassemia. By applying the amino acid score method and its tailored data visualization techniques, a clear profile of protein degradation emerged from the redundant data. Notably, the introduction of heavy labeled peptides or reference samples in targeted quantitative peptidomics led to the first proposal of peptide cluster-based potential diagnostic biomarkers of β-thalassemia. In conjunction with proteomics, the disease characteristics of molecular networks composed of peptides, proteases, and proteins were revealed, highlighting the importance of integrating peptidomics and proteomics technologies.