<p>Disulfide bond linkages play critical roles in maintaining the structural integrity, stability, and biological activity of therapeutic antibodies. Therefore, disulfide bond mapping is considered a critical quality attribute during antibody characterization and development. However, conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based disulfide bond mapping is often complicated by artificial disulfide bond scrambling that occurs during sample preparation and enzymatic digestion, potentially leading to incorrect disulfide bond assignments. In this study, experimental parameters affecting disulfide bond scrambling, including digestion pH, urea, and N-ethylmaleimide (NEM), were systematically evaluated using 20 different digestion conditions followed by LC-MS/MS analysis and data processing. Correctly assigned and scrambled disulfide-linked peptides were identified and quantified based on their relative peak areas. The results demonstrated that the addition of 20 mM NEM effectively suppressed disulfide bond scrambling across all tested pH conditions (pH 6.0–8.0). In contrast, the presence of 8&#xa0;M urea increased disulfide bond scrambling, particularly at higher pH values. A similar but less pronounced pH-dependent increase in scrambling was also observed under buffer-only conditions. Based on the comprehensive evaluation of all experimental conditions, the optimal condition for trastuzumab disulfide bond mapping was determined to include either NEM alone or the combination of urea and NEM, both of which effectively minimized disulfide bond scrambling. These findings provide practical guidance for improving the accuracy and reliability of LC-MS/MS-based disulfide bond mapping and may be broadly applicable to the characterization of therapeutic antibodies.</p>