Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors), biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of Gαi2, Yes- or Src-kinases fused to fluorescent proteins. When expressed in mammalian cells, NANOMS report on loss of membrane anchorage due to chemical or genetic inhibition of myristoylation e.g. by blocking NMT and methionine-aminopeptidase (Met-AP). We used Yes-NANOMS to assess inhibitors of NMT and a cherry-picked compound library of putative Met-AP inhibitors. Thus we successfully confirmed the activity of DDD85646 and fumagillin in our cellular assay. The developed assay is unique in its ability to identify modulators of signaling protein nanoclustering, and is amenable to high throughput screening for chemical or genetic inhibitors of functional membrane anchorage of myristoylated proteins in mammalian cells.