We have developed a methodology using advanced thermal analysis to characterize the role of water in a specially synthesized family of recombinant spider silk-like block copolymers. These proteins were inspired by the genetic sequences found in the dragline silk of Nephila clavipes, comprising an alanine-rich hydrophobic block, A; a glycine-rich hydrophilic block, B; and a C-terminus or a His-tag, H. This family of proteins serves as a model system in which the hydrophobicity is controlled by A and B block lengths, allowing systematic comparison of water effects within the family. Temperature-modulated differential scanning calorimetry and thermogravimetric analyses were employed to capture the glass to rubber transition, T-g, in water-cast protein films. Modeling of the solid and liquid state heat capacity baselines allows us to determine the critical role played by bound water which plasticizes and stabilizes the protein through interchain bonding. In samples containing bound water, two sequential glass transitions, T-g(1) and T-g(2), were observed during heating. The lower temperature glass transition, T-g(1), is related to conformational change induced by bound water removal, the hydrophobicity of the protein sequences, and the crystallinity of the protein. The higher temperature glass transition, T-g(2), is characteristic of the dry protein. The binding energy of water to protein compares favorably to ligandwater binding affinities. The energy absorbed by evaporating water depends upon the volume fraction of the hydrophilic B-block.