Abstract: Polysorbates are a class of hydrophilic non-ionic surfactants characterized by exceptional biocompatibility, rendering them the most frequently employed excipients in therapeutic monoclonal antibodies’ formulations. These compounds play a pivotal role in preventing the formation of antibody aggregates and their subsequent inactivation, while simultaneously mitigating non-specific adsorption onto surfaces of glass vials or intravenous infusion tubing. However, due to inherent variations in synthetic processes and diverse raw material sources, polysorbates exist as complex mixtures with significant batch-to-batch variability in their compositional profiles. Their inherent chemical instability predisposes them to degradation, with degradation products capable of interacting with protein particles present in therapeutic monoclonal antibodies to form insoluble particulates and visible foreign matters. These particulate contaminants pose substantial risks to both drug safety and therapeutic efficacy, thereby underscoring the critical importance of investigating the degradation mechanisms of polysorbates in antibody formulations. Chinese hamster ovary (CHO) cells represent the predominant expression system for antibody production, with trace amounts of host cell protein (HCP) residues persisting during the drug purification process. These trace amounts of HCP residues have been identified as a principal contributor to polysorbate degradation. In this study, a highly sensitive and specific mass spectrometry (MS) method was developed for quantifying trace amounts of polysorbate-degrading esterases in HCP residues. This study aims to elucidate the specific impact of HCP residues on polysorbate degradation, enable comprehensive quantitative analysis of polysorbate-degrading esterases, and thereby provide essential guidance for process optimization and quality control in antibody drug manufacturing. The methodology involved an initial non-targeted screening approach using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF MS) operating under data-dependent acquisition mode. This screening was performed on high-risk HCPs present in Protein A affinity eluates, which yielded comprehensive identification lists of polysorbate-degrading esterases at both the protein and peptide levels. Subsequently, a targeted quantitative approach was established using liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ MS), employing multiple reaction monitoring (MRM) based on characteristic peptide sequences to detect four common polysorbate-degrading esterases: lysosomal phospholipase A2, phospholipase B-like 2, lipase LIPH, and lysosomal acid lipase. The sample processing workflow underwent rigorous optimization, encompassing various parameters such as sample preparation procedures, extraction efficiency, and analytical conditions. The method underwent comprehensive validation, ultimately achieving a limit of quantification of 0.5 μg/g and a limit of detection of 0.1 μg/g. The developed method was successfully applied to quantify these four polysorbate-degrading esterases across various sample types, including purification process samples, bulk drug substances, and final drug products. The results demonstrated that the method exhibits exceptional rapidity, sensitivity, and specificity, making it well-suited for both identification and quantitative analysis of high-risk polysorbate-degrading esterases in therapeutic monoclonal antibodies. This capability provides a robust analytical tool for ensuring drug quality and safety throughout the manufacturing process, offering significant advantages for process development and quality control in antibody drug production.