The manufacturing of biopharmaceuticals and drug substances often involve host cells from different sources. However, host cell proteins (HCP), the proteins released by these cells during the manufacturing process, can negatively impact the stability, quality and efficacy of the final drug product. To ensure the safety of the product, regulatory agencies require extensive monitoring of HCPs during downstream protein purification. The current gold standard of HCP detection is an enzyme-linked immunosorbent assay (ELISA). Albeit its apparent benefits, it has crucial limitations in terms of recognizing different HCPs and providing detailed information on individual HCPs. To address these issues, a proteomics workflow that utilizes multidimensional liquid chromatography and mass spectrometry to accurately quantify and identify HCPs is used. This research provides a more comprehensive understanding of HCPs and their relationship with the protein purification processing steps, which can guide targeted removal strategies and increase overall efficiency. It further is important, as it can help to identify potentially critical HCPs during the manufacturing process, improving the safety and effectiveness of biopharmaceutical manufacturing processes and therefore has the potential to benefit patients globally by ensuring the production of safe and effective drugs.