The alignment of β-sheets within spider dragline silk fibers is an important factor in their tensile strength and extensibility. We are using linear dichroism of the C 1s → π*amide transition measured using scanning transmission X-ray microscopy (STXM) to generate quantitative maps of the
orientation parameters with 30 nm spatial resolution. Here we have extended these measurements from dry samples to samples with partial or full hydration. A device for monitoring and controlling the humidity of a sample in the STXM is described and used to measure the effect of saturated humidity on a section of N. clavipes dragline spider silk. The microstructure and distributions of molecular orientation change considerably with hydration in ways consistent with the supercontraction observed in free standing dragline spider silk. The STXM results are compared to infrared and Raman microscopy results.