Precise regulation of protein assembly at specialized membrane domains is essential for diverse cellular functions including synaptic transmission. However, it remains unclear how protein clustering at plasma membranes is initiated and controlled. Protein palmitoylation, a common reversible lipidation, regulates protein targeting to plasma membranes. Such modified proteins are enriched in these membrane domains. To enable visualization of this dynamic process, we selected a recombinant antibody that specifically recognizes the palmitoylated conformation of PSD-95, a major postsynaptic scaffold protein. When used as a GFP-tagged antibody in living neurons, it revealed that endogenous palmitoylated PSD-95 is partitioned into multiple discrete clusters in a dendritic spine. Membrane-inserted PSD-95 palmitoyltransferase, DHHC2, catalytically generates and maintains these subspine membrane domains, which serve as anchoring positions for AMPA-type glutamate receptors. Thus, the palmitoylating enzyme at plasma membranes creates highly localized hotspots of protein palmitoylation and directly marks sites where specific proteins concentrate to organize membrane domains.