Understanding which proteins are presented at the cell surface is essential for ongoing therapeutic development, and to expand our basic understanding of biology. We envisioned the integration of cell surface engineering with radical-mediated protein biotinylation to profile cell surface proteins (CSPs). This method relies on the pre-functionalization of cells with cholesterol lipid groups, followed by their conjugation with an APEX2 ascorbate peroxidase enzyme. The APEX2 enzyme allows for the covalent conjugation of biotin moieties to nearby residues for subsequent enrichment, and serves as a tool for the enrichment and subsequent MS analysis of CSPs.