Cell surface proteins (CSPs) play a pivotal role in cellular processes and are crucial for differentiating various cell types. Despite the significance of the cell surface-localized proteomeProteomes, it is often characterized using low-throughput techniques that provide limited information from the surface-exposed protein segments of individual cells. If such information is available, it is often scattered across numerous publications, making it time-consuming to gather. To address these challenges, we present a high-throughput method that utilizes biotinylation of accessible primary amino groups within the extracellular regions of CSPs to simultaneously generate several hundred CSP-specific peptides in a single experiment. The steps of the method have been meticulously refined to capture an increasing number of cell type-specific biotinylated peptides localized in cell surface accessible protein regions. The protocol developed is readily applicable to the analysis of both soluble and adherent cell surface proteinsSurface proteins as described in detailed in this paper. Additionally, the method can be used for side-selective protein biotinylation on biological barrier-forming epithelial cells, allowing the qualitative and quantitative characterization of proteins in both their apical and basolateral membranes. The identified amino-modified surface peptides derived from CSPs have potential as targets for various molecular biology research applications, including antibody staining, inhibitor development and monitoring of host–pathogen interactions. Furthermore, label-free quantification of CSPs via mass spectrometry (based on identified peptide list) provides valuable insights into their relative abundance in different cell surface or plasma membrane regions.