Protein palmitoylation describes the post-translational fatty acyl thioesterification of cellular cysteine residues and is critical for the localization, trafficking, and compartmentalization of a large number of membrane proteins. This labile thioester modification facilitates a dynamic acylation cycle that directionally traffics key signaling complexes, receptors, and channels to select membrane compartments. Chemical enrichment and advanced mass spectrometry-based proteomics methods have highlighted a pervasive role for palmitoylation across all eukaryotes, including animals, plants, and parasites. Emerging chemical tools promise to open new avenues to study the enzymes, substrates, and dynamics of this distinct post-translational modification.