Bombyx mori L. silk fibroin (SF) is widely used in different areas due to its ability to form durable and resilient materials with notable mechanical properties. However, in some of these applications the dissolution of SF is required, and this is not often straightforward due to its inability to be dissolved in the majority of common solvents. This work reports a novel approach to dissolve SF using 40 wt % aqueous tetrabutylammonium hydroxide, TBAOH(aq), at mild temperature. A thorough rheological study combined with small-angle X-ray scattering is presented to correlate the SF state in solution with changes in the rheological parameters. The scattering data suggest that the SF conformation in TBAOH(aq) is close to a random coil, possibly having some compact domains linked with flexible random chains. The radius of gyration (Rg) and the molecular weight (Mw) were estimated to be ca. 17.5 nm and 450 kDa, respectively, which are in good agreement with previous works. Nevertheless, a lower Mw value was deduced from rheometry (i.e., 321 kDa) demonstrating a low degree of depolymerization during dissolution in comparison to other harsh processes. The transition from a dilute to a semidilute regime coincides with the estimated critical concentration and is marked by the presence of a shear-thinning behavior in the flow curves, violation of the empirical Cox-Merz rule, and an upward increase in the activation energy. This work paves the way toward the development of advanced high-tech SF-based materials.