Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets.