Exosomes are membranous nanovesicles secreted by cells into the extracellular environment. In the recent years, exosomes have been attributed in intercellular communication via the transfer of biologically active cargo including protein and nucleic acids. Understanding the mechanism of exosomes biogenesis and secretion may provide critical insights as how intercellular communication is orchestrated, especially in pathophysiological settings. A physical coherence and network-based bioinformatics analysis identified 193 candidates as novel regulators of exosomes biogenesis and secretion. NEDD4, a protein implicated in ubiquitin-specific protease activity, was one of the candidates found with significant enrichment score. CRISPR-Cas9 mediated knockout of NEDD4 in human cells validated it as a positive regulator of exosomes biogenesis and secretion. Furthermore, loss of NEDD4 altered the morphology of cells, inducing blebbing and protrusions in the plasma membrane. A significant enrichment of CD63 was observed in the plasma membrane in the absence of NEDD4 as visualized by confocal and electron microscopy. Interestingly, absence of NEDD4 protected colorectal cancer cells from 5-FU mediated apoptosis. Overall, this study implies that NEDD4 plays a novel role in the biogenesis of exosomes and regulating chemoresistance.