Homologous to E6AP Carboxyl Terminus (HECT) E3 ubiquitin ligases are key players in the ubiquitin-signaling pathway that regulates critical cellular activities such as protein localization, viral immune response, and targeted protein degradation. Many researchers currently study the ubiquitylation activity of HECT E3 ubiquitin ligases using gel-based and/or western blot assays, however, these are generally unreproducible and lack the ability to acquire quantitative kinetic data. To address this, we have developed a sensitive FRET-based kinetic assay specific for the HECT E3 ubiquitin ligase family to more precisely investigate the mechanisms of HECT-dependent ubiquitylation. Specifically, we are studying HECT, C2, and WW domain containing E3 ubiquitin protein ligase 2 (HECW2), a poorly studied member of the HECT E3 ubiquitin ligase family that has been linked to numerous genetic diseases. These include, but are not limited to, the accelerated aging disease Hutchinson-Gilford Progeria Syndrome and the epileptic encephalopathy Rett-like Syndrome. Rett-like Syndrome, so named for its phenotypic similarity to Rett Syndrome, has been linked clinically to a number of mutations in the HECT domain of HECW2. Our lab obtained a number of small molecules, identified using a proprietary machine learning algorithm, which are believed to inhibit HECW2 ubiquitylation activity. Our novel assay approach is being used to analyze the effect of these small molecules on wild type HECW2 as well as the disease state mutations of the protein to better understand the effects of the mutations on the protein and potential mechanisms of disease treatment. This assay is also being adapted to examine the kinetic parameters of other members of the HECT E3 ubiquitin ligases, including the HERC5/ISG15 pathway, and will be used in the future screening of other molecules for HECT-dependent diseases.