Abstract: Residual host cell proteins (HCPs) can significantly impact the efficacy and safety of biologics, and are considered as critical quality attributes that require rigorous quality control. Currently, the enzyme-linked immunosorbent assay (ELISA) is the most common methodology for monitoring residual HCPs in biologics. However, the accuracy of ELISA is highly dependent on the specificity of the used antibodies, and it does not provide informations about the types and quantities of individual HCP, making it necessary to complement ELISA with other analytical methods. Mass spectrometry (MS) has emerged as key analytical and characterization technique for HCPs analysis. Without solely depending on antibody affinity recognition, MS enables the identification and quantification of individual HCP, which facilitates a deeper understanding of HCP profiles, clearance patterns, risk assessment, and problematic HCP monitoring. Moreover, the method of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) offers faster development for new products. Despite its advantages, LC-MS/MS faces challenges in HCP analysis, such as the lack of standardized procedures and validation standards, signal suppression in the presence of high-abundance active pharmaceutical ingredient (API), and the high instrumentation cost and specialized expertise barriers in routine quality control settings. Researchers and industry experts are working towards establishing standardized procedures and validation guidelines for HCPs analysis using LC-MS/MS, which includes utilizing data dependent acquisition (DDA) for the construction of project-specific HCP libraries, data independent acquisition (DIA) for efficient HCP screening, and targeted strategy like multiple reaction monitoring (MRM) for absolute quantification of high-risk HCPs. Efforts are also underway to mitigate signal suppression effects and reduce instrumentation costs. To overcome high-abundance API interference in HCP detection, advanced techniques involving API-HCP separation, HCP enrichment, and non-denaturing enzymatic digestion have been developed, allowing for the monitoring of HCPs at exceptionally low levels (0.1 to 0.01 ppm). This paper provided an overview of MS-based HCPs analysis workflow utilized "proteomics" approach, covering essential considerations in instrument selection, sample preparation, LC separation, MS data acquisition, and data analysis and reporting, corresponding optimized practices were discussed. Nowadays ELISA is still a workhorse for residual HCP analysis, however, LC-MS/MS is a valuable tool for the analysis of residual HCPs in biologics. It offers a more comprehensive and detailed view of HCP profiles and their clearance patterns throughout the production process, enabling better risk assessment and monitoring of problematic HCPs. While challenges remain, ongoing research and development efforts are paving the way for the broader adoption of LC-MS/MS in the biopharmaceutical industry, ultimately ensuring the quality and safety of biologic products.