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Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination.We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs.Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions.This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs.