Acyl carrier proteins (ACPs) are key domains in fatty acid and polyketide synthases (PKSs), shuttling intermediates to catalytic partners. Here, we present a protocol for purifying and analyzing ACPs from fatty acid synthases (FASs) and PKSs, using four model ACPs from Mus musculus, Saccharopolyspora erythraea, Streptomyces venezuelae, and Streptomyces hygroscopicus. We describe steps for recombinant ACP production in E. coli; purification via chromatography or precipitation; ACP modification (holo/acyl forms); and analysis using urea PAGE, high-performance liquid chromatography (HPLC), and NMR spectroscopy. For complete details on the use and execution of this protocol, please refer to Gusenda et al.(1).