This study developed a rapid, virus-free method to produce recombinant soluble envelope (sE) proteins of the yellow fever virus (Asibi and 17D strains) and characterized them biophysically as candidate reagents for diagnostics and vaccine research. Flow Induced Dispersion Analysis (FIDA) was used, alongside dynamic light scattering, to assess the hydrodynamic size and monodispersity of the sE proteins, confirming they remained monodisperse and aggregate-free after freeze-thaw and four weeks of storage at −80 °C. It is an example of using FIDA to assess protein sample integrity, monodispersity and stability under storage and handling conditions.