Acid-sensing ion channels (ASICs) are proton-gated sodium channels that contribute to fast synaptic transmission, learning, and memory. ASICs have also gained attention as possible targets to treat ischemic stroke, chronic pain, and neurological disorders. To build the foundation for pharmaceutical targeting, we use chemical biology approaches and fluorometry to better understand the structure-function relationship of these channels. Here, I will present approaches of advanced conventional engineering that help us decipher single subunit contributions in peptide-ion channel interactions and that can incorporate non-canonical amino acids for binding-site identification. Further, I will showcase an intein-mediated protein semi-synthesis approach that allows us to introduce modified sidechains and small organic fluorophores into ASIC1. The results provide an understanding of the role of specific residues in proton sensing and promise insights into the conformational changes of the intracellular domain during gating.