Notch signaling is among one of the highly conserved developmental pathways. Because of its role in the regulation of stem cell self-renewal and cell fate decisions, Notch signaling maintains tissue homeostasis in several organs such as thymus, skin and intestine. The outcome of Notch signaling is tissue and cell-context dependent. However, the underlying mechanisms responsible for this tissue specificity are not fully understood yet. Therefore identification of signaling pathways that are able to modulate Notch signaling will enhance our understanding about the molecular mechanisms responsible for the tissue specific outcome of Notch signaling. In addition, an aberrant activation of Notch signaling is involved in many human cancers such as T-cell acute lymphoblastic leukemia (T-ALL) and breast cancer. The implication of Notch signaling in human cancers highlights the need to develop novel Notch signaling inhibitors as anti-cancer therapies. Therefore the aim of the present study was to identify novel human kinases and chemical compounds that are able to modulate Notch signaling. We have established a DL4:N1 coculture assay system to conduct high-through put screens of chemical compound libraries and of a siRNA library against human kinase genome. The latter screening campaign has led to the identification of PRKC, NRGN and CAMK2B kinases as positive regulators of the Notch pathway. siRNA or pharmacological mediated inhibition of these kinases led to a downregulation of Notch signaling. The screening of chemical compound libraries have led to the identification of three compounds that are able to block Notch signaling activation in in vitro assays. One of these inhibitors termed I3 was further investigated in in vivo studies to determine its effect on Notch-dependent T-cell and Marginal Zone B cell development in mice. Furthermore, the compound I3 has demonstrated a remarkable ability to block tumor progression in xenotransplant models of human T-ALL. In summary, the present study has identified several kinases and chemical compounds able to modulate Notch signalling. These candidates respectively constitute novel molecular targets and lead compounds, that can be exploited to develop targeted anti-cancer therapies.