The quantification of host cell proteins (HCPs) during process development of therapeutics remains an important topic to understand and optimize conditions for their removal. HCPs can often lead to dangerous and deleterious effects on produced therapeutics. Mass spectrometry-based methods can offer high specificity and accurate determination of HCPs quantitation with respect to the standard ELISA based techniques. Herein, we developed a novel HCP quantitation method based on Thermo Fisher Scientific’s SureQuant Targeted Quantitation Assay, a technique that delivers heavy labelled tryptic peptides for targeted quantitation. A panel of 282 peptides was developed to target common and troublesome HCPs in Chinese Hamster Ovary cell lines (CHO), and the panel was subsequently implemented to analyze vaccine and monoclonal antibody therapeutics during process development.