To better understand the effect of mechanical stress during the spinning of silk, the protein orientation and conformation of Bombyx mori regenerated silk fibroin (RSF) films have been studied as a function of deformation in a static mode or in real time by tensile-Raman experiments and polarization modulation infrared linear dichroism (PM-IRLD), respectively. The data show that either for step-by-step or continuous stretching, elongation induces the progressive formation of β-sheets that align along the drawing axis, in particular above a draw ratio of ~2. The formation of β-sheets begins before their alignment during a continuous drawing. Unordered chains were, however, never found to be oriented, which explains the very low level of orientation of the amorphous phase of the natural fiber. Stress-perturbed unordered chains readily convert into β-sheets, the strain-induced transformation following a two-state process. The final level of orientation and β-sheet content are lower than those found in the native fiber, indicating that various parameters have to be optimized in order to implement a spinning process as efficient as the natural one. Finally, during the stress relaxation period in a step-by-step drawing, there is essentially no change of the content and orientation of the β-sheets, suggesting that only unordered structures tend to reorganize.