Abstract: The ability to characterize small molecules that bind to cell surface receptors is a current challenge in drug discovery. Traditional approaches require secondary messenger assays, fluorescent probes, or radioactivity, which are cumbersome, costly, and prone to false positive results. Label-free affinity selection mass spectrometry (ASMS) is a powerful tool in drug discovery to identify small molecule binders, however, it has largely been limited to purified protein and oligonucleotide targets. Here we describe, for the first time, the application of ASMS in a high-throughput workflow to screen for binders of full-length human plasma membrane proteins expressed on the surface of human cells. The methodology combines self-assembled monolayers of alkanethiolates on gold with matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS). The surface chemistry presents a ligand that mediates cell adhesion amongst a background of molecules that prevent non-specific protein adsorption. The monolayer enables the transfection and culturing of living cells in a high-density microarray amenable to automated liquid handling. The cellular arrays are then treated with small molecules either as individual compounds or in pools. After a short incubation, the arrays are washed, dried, and analyzed by MALDI MS. Upon laser activation, the monolayer desorbs and the non-covalent interaction between the small molecule and cellular target dissociates, and the mass ID of the bound ligand is identified. The technology described here opens new avenues for label-free high-throughput screening against a comprehensive library of human cell surface receptors, lead compound off-target screening, and accelerated drug discovery.