Hydration plays a fundamental role in maintaining the three-dimensional structure and function of proteins. In this study, Raman spectroscopy was used to probe the hydration induced structural changes at various sites of lysozyme under isothermal conditions in the range of water contents from 0 to 44 wt %. Raman hydration curves were constructed from detailed analysis of marker bands. Transition inflection points (w(m)) and onsets determined from the hydration curves have shown that structural changes start at 7-10 and end at about 35 wt % water. The onset of structural changes coincides with the onset of the broad glass transition earlier observed in this system. The increase of α-helix content starts at very low concentrations of water with w(m) = 12 wt %. Monitoring the development of importance for enzymatic action hydrophobic clusters has revealed wm = 15 wt % and completion of the process at 25 wt %. The parameters of 621 cm(-1) (Phe) and 1448 cm(-1) (CH2 bending) modes were found to be sensitive to hydration, suggesting changes in organization of water molecules near the protein surface. The native structure of lysozyme was achieved at 35 wt % water where its content is high enough for filling the space between lysozyme molecules.