As a selective permeability interface, the blood-brain barrier (BBB) restricts drug access to the brain, complicating theranostic strategies for neurological disorders. The selective permeability of the BBB is primarily controlled by brain microvascular endothelial cells (BMECs). Cell surface proteins can be endocytosed into brain microvascular endothelial cells, serving as promising candidates for receptor-mediated transcytosis across the BBB. However, to date, systematic analysis of the internalization of surface proteins in human BBB models still has some limitations. In this study, we employed high-resolution mass spectrometry (HRMS) based on Data-Independent Acquisition (DIA) combined with cell surface biotinylation to comprehensively identify internalized surface proteins in human brain microvascular endothelial cells (HBEC-5i). This approach overcomes traditional proteomics limitations, enabling high-throughput quantification of hundreds of surface proteins. Our analysis identified 232 internalized cell-surface proteins, with 54 showing HBEC-5i specificity compared to dermal (HMEC-1) and pulmonary (HULEC-5a) microvascular endothelial cells. Through integration with mRNA expression data from public databases and immunohistochemical evidence from the Human Protein Atlas, we identified Plastin 3 (PLS3) and Collectin Subfamily Member 12 (COLEC12) as key candidate proteins. Both proteins exhibited elevated expression and distinct plasma membrane localization patterns in HBEC-5i cells. Our study highlights the unique advantages of DIA high-resolution mass spectrometry in profiling BBB-related proteins and provides valuable insights for developing targeted strategies to enhance central nervous system drug delivery.