Spider silk has incredible mechanical properties which are sought after for countless applications; however, collecting silk on an industrial scale is not practical and synthetic silks have not yet achieved the desirable qualities of the native material. The aim of this work is to understand how both the silk protein sequence and spinning conditions contribute to the qualities of the fiber, with the goal of applying this knowledge to design mechanically tunable synthetic silks. The hierarchical organization of the starting material, an intrinsically disordered, concentrated protein hydrogel (dope), isolated from L. hesperus (black widow) spiders was investigated using solution NMR, DLS, negative stain TEM, and iSCAT. Dimerization of silk proteins was observed with iSCAT, indicating disulfide bonds form between protein terminal domains. TEM imaging of native silk prior to fiber formation revealed two populations of micelles at 50 and 300 nm, pointing to hierarchical organization at the nanoscale. Phosphate-induced liquid-liquid phase separation (LLPS) was induced both in recombinant and native major ampullate (MA) silk dopes and studied with solution NMR, focusing on Tyr and Arg residues. The largest chemical shift perturbations were observed in the Arg amide and other polar residue side chain sites. The most significant backbone dynamic differences were found in the GGX and GAA motifs, which form the interface between crystalline poly(Ala) domains and disordered Gly-rich regions in fibers. This indicates pre-ordering of specific repetitive regions en route to β-sheet formation. Further, two distinct silk spinning dopes from orb weavers, MA and aciniform (AC) silk isolated from Trichonephila and A. aurantia, were investigated with NMR. While some differences were observed in AC silks indicating the presence of α-helical structure, all silks were found to be predominantly random coil in solution. Finally, elemental analysis was conducted on silk samples using ICP-MS to probe whether spiders and silkworms utilize similar chemistries to initiate LLPS pre-ordering in silk spinning. Both Trichonephila MA and B. mori silkworm silks were found to undergo phosphate-induced LLPS, which is hypothesized to be an important intermediate step in silk fiber formation and critical to biomimetic spinning of synthetic silks with native-like mechanical properties.