In humans, many Notch mutations in the NRR have been reported in both solid tumours (Aster et al., 2017; Mutvei et al., 2015; Zhang et al., 2016) and blood cell cancers (Rosati et al., 2018; South et al., 2012), such as T-ALL (Belver and Ferrando, 2016; Pear and Aster, 2004). Here, we generated several mutations located in different regions of the NRR of Drosophila Notch to investigate their consequences on Notch activation and compare their behaviour to previously studied T-ALL mutations. The latter included perturbation of a putative dimerisation interface that was previously identified through crystallographic studies of human Notch (Gordon et al., 2009; Xu et al., 2015). Mutations of this region in human NOTCH3 increased ligand-independent basal Notch activation (Xu et al., 2015). However, S2 cells lack some cofactors, including Fringe, specific DSL ligands, and specialised cell junctions, that influence Notch activation.