The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17, also known as TNFα convertase, TACE) serves as a critical line of defense against injury and infection. ADAM17 maintains the skin and intestinal barrier by processing several ligands of the Epidermal Growth Factor Receptor and regulates innate immunity by releasing the pro-inflammatory cytokine TNFα. ADAM17 can be rapidly activated within minutes by a number of different signaling pathways and previous studies demonstrated the transmembrane domain of ADAM17 is required for its activation. The seven-membrane spanning inactive Rhomboids 1 and 2 (iRhom1/2) were discovered to be regulatory cofactors of ADAM17 proteolysis and iRhom2 has emerged as a novel therapeutic target for TNFα-dependent inflammation. Moreover, a point mutation in the first transmembrane domain of iRhom2, termed sinecure, also results in a loss of function in ADAM17. Here, I describe a structure-function analysis that I performed to explore the role of the transmembrane domains (TMD) of ADAM17 and iRhoms in modulating the activity of ADAM17. This work also provided the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 are important for regulating the stimulation and substrate selectivity of ADAM17. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2. In addition, I uncovered regions in iRhom2 and ADAM17 that are important for the regulation of ADAM17-dependent constitutive activity. Finally, I developed and validated several robust cell-based assays for high throughput screening of small molecule inhibitors to ADAM17/iRhom2-dependent activity. Taken together, this work provides novel mechanistic and functional insights into how iRhom2 regulates ADAM17 and will aid the development of inhibitors of inflammation and auto-immune diseases such as Rheumatoid Arthritis.