Host cell proteins (HCPs) co-expressed during the production of biotherapeutics can affect the safety, efficacy and stability of the final product. As such, monitoring HCPs populations and amounts throughout the production and purification process are an essential part of the overall quality control framework. Mass spectrometry (MS)is used as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for simultaneous identification and quantification of HCPs, particularly for the analysis of downstream processes. In this study, we present an MS-based analytical protocol with improvements in both speed and identification performance that can be implemented for routine analysis to support upstream process development. The protocol adopts a streamlined sample preparation strategy combined with a high-throughput MS analysis pipeline. The developed method identifies and quantifies over 1000 HCPs, including 20 proteins listed as high risk in literature, in a clarified cell culture sample with repeatability and precision shown for digest replicates. In addition, we explore the effects of varying standard spike-ins and changes to the data processing pipeline on absolute quantification estimates of the HCPs, which highlights the importance of standardization for the wider use in industry.