Exosomes are small membrane extracellular vesicles that are secreted under physiological and pathological conditions. However, very little is known about the biogenesis of exosomes. Here, we studied ESCRT dependent mechanism of exosomal biogenesis by physical coherence and network based analysis. Using a conserved interaction network and physical coherence model, we indented to identify novel regulators of exosome secretion. To remove literature bias on the protein-protein interaction models, we performed physical interaction enrichment analysis and identified ESCRT protein neighbors that could regulate exosomal biogenesis. A total of more than 50 proteins including NEDD4, SDCBP and STAMBP with significant p-values were identified. SDCBP has already been implicated in exosomal biogenesis in previous studies and hence validates our approach. Next, we evaluated the role of NEDD4 and STAMBP in exosomal biogenesis by molecular biology and biochemical experiments. In this study, we investigated the role of the E3 ligase NEDD4 in the biogenesis of exosomes. To address this, we generated CRISPR based NEDD4 knockout (KO) in LIM1215 colorectal cancer cells. In addition, we utilised MEFs from Nedd4 KO mice. Exosome were isolated from wild type (WT) and KO cells using differential centrifugation coupled with ultracentrifugation. The isolated exosomes were quantified based on nanoparticle tracking analysis (NTA) and total protein amount. The analysis revealed significant reduction in exosome secretion in NEDD4 KO cells compared to WT cells suggesting that NEDD4 is a novel regulator of exosomal biogenesis. Furthermore, immunoblotting was performed to confirm the reduced levels of exosomal enriched markers such as Alix and TSG101. Follow up quantitative proteomic analysis of exosomes derived from WT/KO cells revealed protein cargo dependent on NEDD4. Hence, the results validate the predictions based on physical coherence and network models. Overall, in this study we have identified novel regulators of exosomal biogenesis using an integrated bioinformatics and experimental approach.