Even at low levels, these residual proteins may trigger immune responses or impact drug stability, making their removal essential. To ensure product safety and efficacy, manufacturers must identify, monitor, and minimize HCP contamination throughout production. Host cell proteins (HCPs) are process-related impurities derived from host organisms used for recombinant protein production in biopharmaceutical manufacturing. The generation of HCPs may lead to potential safety risks, such as immunogenicity, reduced drug efficacy and long-term side effects. It provides best practices for measuring residual host cell proteins (HCPs) in biologics using liquid chromatography–mass spectrometry (LC-MS/MS). The chapter supports regulatory compliance by offering detailed guidance on method validation, system suitability, and quantitation of individual HCPs. Cell lines are essential for developing and manufacturing biopharmaceuticals and viral vectors, but all cell line expression results in contamination by residual host cell proteins (HCPs). The issue of residues of host cell proteins (HCPs) plays an important role in the manufacture of peptide products. In July, the FDA called for comments via the Federal Register to help assess the immunogenicity risk of such HCPs. As product development progresses, the level of host cell material should be further reduced, as technically and logistically feasible. Most protein therapeutics are manufactured in nonhuman host cells; therefore, one of the major concerns of the presence of residual HCPs is the potential for any one or more of the HCPs to elicit an immunogenic response. USP General Chapter 1132 provides a comprehensive, informational framework for controlling residual host cell proteins (HCPs)—process-related impurities from the production cell line (e.g., CHO, E. coli). Integrated solutions leveraging real-time PCR or dPCR technologies are necessary to meet regulatory needs. This article will cover some of the latest regulations around residual DNA amounts in the product, as well as quantitating host cell and pDNA, and the presence and size of the E1A oncogene. The Food and Drug Administration (FDA or Agency) is establishing a public docket to collect information and comments on evaluating and mitigating the immunogenicity risk of host cell proteins (HCPs).