Properties of polyhydroxycompounds, such as sugars and polyols, which are used to stabilize various pharmaceuticals and biological objects during freezing, desiccation, and storage, depend on water content. In this investigation, glycerol-water mixtures containing 40 to 0 wt% water are studied by FTIR spectroscopy and molecular dynamics simulation. The HOH bending (approx. 1640 cm−1) band in the FTIR spectra is essentially independent of water content in the range of 0 to approx. 12 wt% water, whereas a red shift to lower wavenumbers is observed as water content increases beyond 12 wt%. Water clustering patterns, which obtained from MD simulations, show a gradual decrease of unclustered water molecules at a higher water concentration, with the corresponding increase in small (3–4 water molecules) water clusters. Large water clusters (>30 molecules) appear at 9 to 12 wt% water; at 25 wt% water, smaller water clusters disappear, with essentially all water molecules belonging to continuous water domain. The water clustering and hydrogen bonding patterns are discussed in view of fundamental physical chemical processes including proton transfer, water catalysis of chemical reactions, and freezing behavior.