The formation of the nervous system relies on a crucial process called axon pathfinding. This process involves the precise growth and connection of axons to their appropriate target site signaled by guidance cue ligands such as Netrin-1, Ephrin, Semaphorin and Slit. The growth cones, at the end of axons, sense guidance cues and mediate axonal growth and direction. Net-1 can attract or repel growth cones through binding to transmembrane receptors, Uncoordinated family member 5 (UNC5), Deleted in Colorectal Cancer (DCC) and Neogenin (NEO). Net-1-receptor complexes trigger intracellular signaling cascades that direct axon trajectories towards Net-1 through DCC or NEO, and away from it through UNC5 dependent/independent DCC. Beyond axon guidance, Net-1 receptors also function as dependence receptors. Overexpression of Net-1 acts as a survival factor and inhibiting UNC5 and DCC mediated apoptosis in various cancers. Inhibition of Net-1 interactions with its receptor has been shown to trigger tumor cell death, making it a potential therapeutic target. This study presents a comprehensive biophysical and structural examination of the highly dynamic Net-1 in complex with UNC5B. The objective is to elucidate novel insights into this interaction and identify potential molecular binding partners. A combination of in solution biophysical techniques including size-exclusion chromatography (SEC), SEC coupled multi-angle light scattering (SEC-MALS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS) were used to characterize Net-1 alone and in complex. The biophysical and structural analyses of Net-1-UNC5B complex, and Net-1-anti-Net-1 antibody further elucidated the mechanism of Net-1-UNC5B interaction, and the mode of inhibition by the antibody as a therapeutic strategy for cancer. Finally, we found a direct interaction between Net-1 and Robo2 that suggests a novel mechanism which connects Net-1 and Slit signaling pathways. Using biophysical and structural techniques, Net-1 was found to interact with Robo-2 in a similar manner as UNC5B. This finding can highlight the molecular mechanism of the DCC-Robo heterodimerization in commissural axon guidance where Net-1 and Slit signaling pathways are coordinated. Collectively, the biophysical and structural insights presented in this study regarding Net-1 interactions with its receptors can guide the design of specific inhibitors to disrupt Net-1-receptor complexes.