Bruton’s Tyrosine Kinase (BTK) is a member of the Tec family non-receptor kinases and is an essential component of the B-cell signaling cascade, contributing to both B-cell development and antibody production. BTK is of clinical importance as it is the target of Ibrutinib, which is used for treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Mutations in the BTK gene also cause a genetic immunodeficiency disorder, known as X-Linked Agammaglobulinemia (XLA). There are numerous XLA mutations identified throughout the BTK enzyme, but our attention focuses on a cluster of mutations residing in the BTK SH2 domain that we hypothesize is part of a regulatory interface that positively influences kinase activity. To investigate this further, we employed sortase-mediated ligation (SML) to generate a segmentally isotopic labeled protein that was analyzed using solution nuclear magnetic resonance (NMR) spectroscopy. The partially labeled protein allowed us to identify an intradomain regulatory interaction between the BTK SH2 and catalytic kinase domain. Further, the BTK SH2-Kinase protein was studied using an NMR technique known as paramagnetic relaxation enhancement (PRE) which informs on surface exposed amino acid residues, aiding the identification of a potential intramolecular interface. From the NMR data, an activation-state model of BTK has been constructed using computational modeling and we are currently testing this model using mutagenesis and coupled-kinase activity assays. We anticipate that interrogation of the SH2-Kinase interface will elucidate the molecular mechanisms that govern BTK activation, a phenomenon that is less well understood for Tec family kinases. These efforts contribute to the development of drug therapies for modulating kinase activity within the immune cell.