Coenzyme A (CoA) analogues are widely used as tools in chemical biology. Their applications include being used as inhibitors and mechanistic probes of CoA-dependent enzymes, and in the labeling of especially carrier proteins active in polyketide and nonribosomal peptide synthesis with fluorescent probes or affinity tags, or with chemically reactive cross-linking moieties. The preparation of CoA analogues has been simplified tremendously since the discovery of the CoA biosynthetic enzymes. Consequently a large number of such analogues have been prepared in recent years by the biotransformation of pantothenamide precursors (a bottom-up approach) or by aminolysis of activated pre-CoA thioesters (a top-down approach). This Minireview provides an analysis of the biosynthetic enzymes used in the biocatalytic production of CoA analogues, an overview of the different ways in which they are currently applied and a description of the types of CoA analogues that are currently easily accessed by such methods.