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A nanoliter liquid chromatography–high resolution mass spectrometry-based method was developed for quantitative proteomics analysis of COVID-19 vaccines. It can be used for simultaneous qualitative and quantitative analysis of target proteins and host cell proteins (HCPs) in vaccine samples. This approach can directly provide protein information at the molecular level. Based on this, the proteomes of 15 batches of COVID-19 inactivated vaccine samples from two companies and 12 batches of COVID-19 recombinant protein vaccine samples from one company were successfully analyzed, which provided a significant amount of valuable information. Samples produced in different batches or by different companies can be systematically contrasted in this way, offering powerful supplements for existing quality standards. This strategy paves the way for profiling proteomics in complex samples and provides a novel perspective on the quality evaluation of bio-macromolecular drugs.