Cellular DNA is constantly subjected to damage from both internal and external sources. These damaging agents give rise to various types of lesions, with the most severe being the DNA double-strand break (DSB), where both strands of the DNA molecule are severed, resulting in complete separation DNA molecule. DNA lesions present in S phase can impede the progression of replication forks causing them to stall in a process known as replication stress. Persistently stalled forks are prone to collapse resulting in the formation of a single ended DSB. To overcome the challenge imposed by DNA lesions, mammalian cells have developed complex repair pathways designed to resolve these lesions while simultaneously balancing accuracy and speed. The Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of Serine/Threonine kinases that coordinate various processes to maintain genome stability. Three of the members of this kinase family, ATM, ATR, and DNA-PKcs, are master regulators of the responses DNA damage and replication stress. Although previous studies have identified direct targets of these three kinases, we still lack a comprehensive understanding of signaling specific to each kinase. Moreover, phosphoproteomic studies intent on observing individual kinase signaling have been hindered by their overlapping function. I applied a quantitative phosphoproteomic approach to extensively investigate DNA-PKcs dependent signaling in response to treatment with ionizing radiation and replication stress inducing drugs. My analysis revealed a novel motif that is directly phosphorylated by DNA-PKcs expanding the repertoire of known targets of the kinase. Interestingly, these novel substrates are enriched in proteins involved in RNA biogenesis and processing, with one of the identified targets being the splicing factor SF3A3. Furthermore, I show that DNA-PKcs mediated phosphorylation of SF3A3 plays a crucial role in governing the dynamics of replication forks during replication stress. Overall, my findings provide valuable insights into the specific signaling pathways governed by DNA-PKcs and highlights the intricate connections between DNA damage response, RNA processing, and replication stress.