The characterization of root vegetables’ bioactive proteins biophysically and biochemically becomes imperative as they play an incredibly important role in the discovery, development, and formulation of bioactive proteins as biopharmaceutical products. This is because bioactive proteins differ in terms of size, molecular weights, structures, and physicochemical properties. Biophysical and biochemical characterization employs several techniques ranging from simple to complex procedures to give insight into proteins’ high-order structures, functions, and biochemical activities. Owing to the increasing awareness and acceptance of the use of peptides and proteins of root vegetable origin as treatment agents against some debilitatingly chronic diseases, researchers are now exploring an eco-innovative approach to reduce their loss by getting to structurally and functionally characterizing them. Several biophysical and biochemical tools are employed routinely for protein characterization and some of which are ultraviolet-visual (UV-Vis) spectroscopy, high-performance liquid chromatography (HPLC), circular dichroism (CD), intrinsic tryptophan fluorescence (ITF), differential scanning calorimetry (DSC), thermal shift assay (TSA), among others.