We developed a novel strategy using integrated DDA-PRM-dMRM to comprehensively profile and quantify high-risk HCPs in CHO cell-derived biotherapeutics. Firstly, we performed deep proteomic analysis of CHO cells using DDA-PASEF mode, constructing a high-quality spectral library covering all potential HCPs. Based on the constructed library, we validated the target list using PRM-PASEF and MRM modes.