This dissertation presents the results of nonlinear spectroscopic studies with the goal to better understand the structure of water around DNA. The flexibility, function, and structure of DNA are partially dependent on the layers of surrounding water that hydrogen-bond to DNA. The first part of this dissertation describes chiral sum frequency generation spectroscopy (SFG) as a label-free, surface-specific, chiral-sensitive, nonlinear vibrational spectroscopy. By controlling the frequency and polarization of the beams involved in SFG experiments, chiral study of biomolecules and the water around them is possible. The second part of this dissertation develops a robust method for chiral SFG using polarization multiplexing and self-referencing. The simple combination of a polarizer, achromatic waveplate, and beam displacer precisely controls the polarization of detected light. Through demonstration on archetypal achiral and chiral samples, the method is shown to increase signal-to-noise ratio, reduce detection time, and provide robustness to both interference and pure chiral SFG experiments. The third part of this dissertation describes the application of the newly developed chiral SFG method to the water around DNA. A copper-free click reaction was adapted to bond DNA strands to visible- and infrared-transparent prisms. Two 24-base pair sequences of DNA—alternating thymines and adenines and alternating guanines and cytosines—were studied dry and wet using chiral SFG. The nonzero chiral response across the OH stretch region indicated the heterogeneous structure of water around DNA. Waters bound strongly to DNA, waters hydrogen-bound to other waters, and waters with extremely weak hydrogen-bonding all appear in the chiral SFG spectrum. These results confirm the presence of a strongly bound minor groove spine of hydration, but they also could support “soft” waters in the major groove that are affected by the chirality of the DNA structure, but do not bind directly to the DNA. There is additional evidence of non-hydrogen bonding waters that may be facing non-polar structures in DNA. The vibrational spectroscopic access to only the waters around DNA in an in situ, room temperature experiment is unprecedented. Future work is discussed that capitalizes on the advances shown in this dissertation.