Notch is an evolutionarily conserved pathway involved in many fundamental biological processes in living organisms. It has been reported previously that Notch is modulated through a fine and intricate regulatory network including Notch endocytic trafficking. In Drosophila, Such endocytic regulation acts to maintain the stable output of Notch signalling in response to different environmental variants such as temperature. Intracellular Ankyrin domain of Notch is a highly-conserved region for critical protein interactions, such as with Deltex, which is a key regulator of Notch endocytic pathway. Mutations in this region may lead to severe diseases and cancer-related mutations have been reported in Human Notch orthologs. This project aims to understand how mutations in this region affect Notch signalling and trafficking. In addition, by comparing both in vitro and in vivo data, we attempt to get some clues of the link between phenotype and genotype of these mutations. Starting from a Drosophila mutation called Su42c, we found that Su42c reduces Notch endocytic signalling itself. In addition, in concert with an Abruptex mutation 9B2, Su42c completely abrogates Notch endocytic signalling. These changes could be attributed to the biochemical and endosomal sorting changes deriving from Su42c and 9B2. We also functionally analysed some cancer-related mutations that were transferred to Drosophila Notch and found that these mutations affect Notch through varied mechanisms. Finally, we attempted to establish a universal method of CRISPR to put in any ANK mutation of interest in Drosophila. This project shows new insights into Notch endocytic regulation.