Protein S-acylation, an enzymatically modulated lipid post-translational modification, is frequent and consequential; at the cellular level, it regulates protein subcellular trafficking and signaling activity, and at the organismal level, has been implicated in cancer and neurologic and inflammatory disease. However, the impact of dynamic S-acylation for many proteins remains unknown, in part due to lack of inhibitors for the enzymes that catalyze lipid addition, the DHHC family proteins. Not only is the most commonly used pan-DHHC inhibitor, 2-bromopalmitate (2BP), toxic and poorly potent, it also concurrently inhibits the S-acylation eraser proteins, the acyl protein thioesterases (APTs). Here, we report the synthesis and characterization of CMA a DHHC inhibitor with improved potency and selectivity. We then use it to establish the dynamics and functional consequences of S-acylation for a previously undescribed acylated protein, the extracellular signal-regulated kinase (ERK1/2), whose S¬-acylation is resistant to current DHHC inhibitors. As an α-halo fatty acid, 2BP reacts with a range of nucleophilic amino acids and forms reactive acyl CoA intermediates in cellulo. We therefore proposed to exchange the α-halo with an acrylamide, a warhead known to react with cysteine over serine residues that will not undergo metabolic conversion. Synthesis and screening of a panel of acrylamide-containing lipids revealed CMA as a potent DHHC20 inhibitor in vitro, with the ability to significantly decease both substrate and global protein S-acylation in cellulo. Competitive activity-based protein profiling (ABPP) demonstrated that SA8 engages directly with a panel of DHHCs in cells. Moreover, CMA is less toxic than 2BP, does not inhibit eraser APT activity, and is able to effect cellular responses previously reported to result from loss of S-acylation. These results position the acrylamide as an inhibitory scaffold for DHHC family proteins. We next CMA to probe the effects of perturbing palmitoylation on epidermal growth factor (EGF)-triggered signal transduction. Characterization of disruptions along this signaling cascade enabled the identification of a previously undescribed acylated protein, the extracellular signal-regulated kinase (ERK1/2), whose S-acylation is resistant to 2BP but responsive to CMA. We describe the molecular determinants of ERK1/2 acylation and cross-talk between ERK1/2 phosphorylation and palmitoylation, suggesting the criticality of S-acylation in regulating the activation and downstream behavior of this key effector protein. We then conduct a structure-activity study of CMA, which demonstrates the criticality of the lipid tail and the potential of scaffold extension, but underscores the need for novel inhibitors. To meet this need, we design and validate a fluorescence-based high-throughput assay for DHHC activity in vitro using a panel of fluorogenic probes. These palmitoyl-CoA mimetic probes are recognized and cleaved by the DHHC family proteins, providing a direct, sensitive, and simple readout of zDHHC2, 3, and 20 activities. After establishing its suitability for a high throughput screen, we screen a library of ~1,000 molecules against zDHHC20, identifying new acrylamide-based inhibitory scaffolds for zDHHC20. In sum, this work reveals the potential of acrylamide-based DHHC family inhibitors, introduces a novel platform for the identification of new inhibitors, and emphasizes that much remains unknown about the significance of dynamic S-acylation in regulating protein activity and cellular events.