Although the post-translational modification (PTM) AMPylation, or adenylylation, was discovered in bacteria over 40 years ago, it was not until the past decade that AMPylation in metazoans was reported. Due to the relatively recent finding of AMPylation in metazoan, not many substrates are known that undergo AMPylation and how this PTM regulates these proteins. We developed an AMPylation recognition technique by utilizing a catalytically inactive AMP binding domain, to identify new substrates and to study the regulative role of AMPylation. To understand the recognition mechanism, we solved the structure of the AMP binding domain bound to AMPylated GlnA using Cryo-EM, the first ever AMPylated substrate found. Using our structure and homolog sequences to guide the mutagenesis, we optimized our system for various applications including the recognition of AMPylation by far western blots, enrichment of AMPylated substrates for proteomics, and purification of AMPylated substrates for biochemical and biophysical studies.