The Hedgehog (Hh) signaling pathway is an essential player in vertebrate embryotic organ development, controlling the formation of nearly every organ in our bodies. Insufficient Hh pathway activity during development results in birth defects, while improper activation postnatally is associated with many cancers including basal cell carcinoma and medulloblastoma. Smoothened (SMO) is an atypical G protein-coupled receptor that is integral to Hh signal transduction. SMO signals intracellularly by directly binding to the protein kinase A catalytic subunit (PKA-C), sequestering it at the membrane and inhibiting its downstream enzymatic activity. The biochemical and structural basis for the SMO-PKA interaction remains largely unknown. Analysis of this protein interaction via crystallography or cryogenic electron microscopy remains difficult due to the unstructured nature of the SMO cytosolic tail. Information regarding the structural basis for how the SMO cytosolic tail binds with PKA-C can be improved upon using nuclear magnetic resonance (NMR) spectroscopy. An optimized expression protocol was used to purify the SMO cytosolic tail, and then biochemically modify the protein for structural studies. Specifically, a covalently attached nitroxide spin label, methanethiolsulfonate (MTSL) was added to various positions in the SMO cytosolic tail to enable paramagnetic relaxation enhancement (PRE)-NMR studies with isotopically labeled PKA-C. The information provided from the PRE-NMR study uncovers a better picture of the binding interaction between SMO and PKA-C. Understanding the structural basis for the SMO-PKA complex can provide critical insights into an essential step in Hh signal transduction and may lead to more effective therapeutic agents to treat a range of different cancers.