The development of instruments combining multiple characterization and imaging tools drove huge advances in material science, engineering, biology, and other related fields. Notably, the coupling of SEM with micro-Raman spectrometry (μRaman) provides the means for the correlation between structural and physicochemical properties at the surface, while dual focused ion beam (FIB)-scanning electron microscopes (SEMs) operating under cryogenic conditions (cryo-FIB-SEM) allow for the analysis of the ultrastructure of materials in situ and in their native environment. In cryo-FIB-SEM, rapid and efficient methods for assessing vitrification conditions in situ are required for the accurate investigation of the original structure of hydrated samples. This work reports for the first time the use of a cryo-FIB-SEM-μRaman instrument to efficiently assess the accuracy of cryo-fixation methods. Analyses were performed on plunge-freezed highly hydrated calcium phosphate cement (CPC) and a gelatin composite. By making a trench of a defined thickness with FIB, μRaman analyses were carried out at a specific depth within the frozen material. Results show that the μRaman signal is sensitive to the changes in the molecular structures of the aqueous phase and can be used to examine the depth of vitreous ice in frozen samples. The method presented in this work provides a reliable way to avoid imaging artifacts in cryo-FIB-SEM that are related to cryo-fixation and therefore constitutes great interest in the study of vitreous materials exhibiting high water content, regardless of the sample preparation method (i.e., by HPF, plunge freezing, and so on).