Host cell proteins (HCPs), which are biologic drug product impurities released during cell growth and subsequent processing, can detrimentally affect final drug product safety and efficacy, therefore must be removed post-harvest through a series of purification steps. A scalable platform process for mAb purification was established, and three intermediate product samples were used in this study for LC-MS/MS based HCP analysis using the new HCP analysis workflow in Thermo Scientific™ BioPharma Finder™ 4.1 Software. The goal of this study was to gain additional knowledge on influencing factors of upstream processing (USP) and downstream processing (DSP) into individual HCP clearance in recombinant mAb products, thus supporting decisions to be made on the most suitable HCP control strategies. Here we demonstrated that the optimized non-denaturing protein digestion facilitates detection of HCPs. Removal of undigested protein with heat-treatment prior to MS analysis reduces the dynamic range of the sample, increasing the probability of HCP identification. 676 HCPs (total HCP 49473.27ppm) in protein A pool sample, 111 HCPs (total HCP 1253.38ppm) in AEX pool sample and 7 HCPs (total HCP 89.66ppm) in CEX pool sample were identified and quantified with at least 3 unique peptides present. The decreasing trend of number of detected HCPs provided clear evidence of step-by-step HCP clearance. Several high-risk HCPs were selected for monitoring in all samples during process development. 30 high-risk HCPs were detected and quantified in protein A pool sample, which indicated that these HCPs may be co-purified with mAb at Protein A affinity step. However, only 15 high-risk HCPs were detected in the AEX pool and 3 in the CEX pool sample. The developed method can effectively support biologics downstream process development by focusing on individual HCP clearance at each step and allows for the better process understanding and rapid process improvements throughout the drug product lifecycle.