Spider silk's mechanical properties make it an interesting material for many industrial applications. The structure and nanoscopic organization of its proteins are the basis of these qualities. In this study, the emission maxima of the autofluorescence from the protein core of major and minor ampullate silk fibers from the orb-web-weaving spider Nephila madagascariensis are determined and found to be 534 ± 11 and 547 ± 19 nm, respectively. Molecular conformational changes during applied strain are observed in both fiber types using two-photon excitation polarization measurements. Our findings showed that within the fibers the autofluorescent dipoles are separated into two distinct populations, one randomly orientated (amorphous regions) and one with aligned dipoles as found in crystalline structures. The crystalline-amorphous ratio was determined, and it was found that the crystalline dipoles made up around 30 and 20% of the autofluorescent dipoles in major and minor ampullate silk fibers, respectively. Using two-photon polarization measurements, it is possible to directly observe that the major and minor ampullate silk fibers structurally adapt to the applied stress, as well as discern different molecular conformational changes between major and minor ampullates. It was seen that the crystalline-amorphous ratio increased, with up to 9% for major fibers and 6% for minor fibers, as strain was applied, suggesting a conformational adaptation of the fiber, interpreted as noncrystalline 310-helices transforming into crystalline β-sheets.