Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design. Structural studies suggest that proteases from Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) exist in open and closed conformations. Solution NMR studies showed that the closed conformation is predominant in solution and should be utilized in structure-based drug design. Here, we reviewed solution NMR studies of the proteases from these viruses. The accumulated studies demonstrated that NMR spectroscopy provides additional information to understand conformational changes of these proteases in the absence and presence of substrates/inhibitors. In addition, NMR spectroscopy can be used for identifying fragment hits that can be further developed into potent protease inhibitors.