Modern MS-based proteomics provides an opportunity to systematically characterize proteins. However, due to the complexity of cell-surface proteins, the labor-intensive workflow, and the limit of clinical samples, comprehensive characterization of the surfaceome remains extraordinarily challenging, especially in clinical studies. Developing and optimizing surfaceome enrichment methods and utilizing automated sample preparation workflow can expand the applications of surfaceome analysis and deepen our understanding of the functions of cell-surface proteins.