Comprehensive profiling of the cell surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here, we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step that we called plasma membrane-BioID. The lipid modified BirA* enzyme (MyrPalm BirA*) was targeted to the plasma membrane where it effectively biotinylates plasma membrane proteins. Biotinylated proteins were then affinity purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and plasma membrane-BioID is ideal to overcome the inherent limitations of identification of integral membrane proteins and yields highly pure plasma components for downstream proteomic analysis.