The plasma membrane proteome is highly dynamic and has crucial roles in many biological processes rendering this subproteome a prominent target for pharmaceutical intervention. Furthermore, there is growing evidence for active drug in- and efflux mechanisms by membrane-bound transporters. Thus, expression of these proteins affects drug-target engagement and can lead to drug resistance. A detailed understanding of the plasma membrane proteome composition and its dynamics can have a significant impact on drug discovery. However, analysis of the cell surface proteome is challenging due to its biochemical properties and low abundance. Here, a sensitive and selective enrichment protocol was established for the comprehensive identification and quantification of cell surface accessible proteins by mass spectrometry. In addition, we combined the recently described thermal proteome profiling (Savitski 2014) enabling target identification and determination of target engagement with our protocol to specifically monitor drug binding to cell surface presented membrane proteins on live cells.