Alzheimer’s Disease (AD) is the main form of dementia, currently affecting more than 55 million people worldwide. There is a pressing clinical need for a definitive diagnostic test and effective therapy for AD. Light-based label-free techniques are particularly promising as they provide 3D imaging capabilities with chemical specificity and subcellular resolution. Vibrational spectroscopy methods have reached a level of advancement which enables translational medicine and potential for diagnosis and therapy. Our previous work has applied Fourier Transform Infrared (FTIR) imaging, Raman and Brillouin microscopy to the study of amyloidopathy in ex vivo brain tissue from an animal model of disease, providing an insight into the chemical and mechanical properties of amyloid beta plaques. The present work extends the analysis to an animal model of tauopathy (rTg4510 mice) to investigate the variations of molecular composition in the brain hippocampus (CA1 region) between transgenic (TG) and wild-type (WT) littermate control mice. In the study, combining multiple advanced label-free spectroscopic methods based on Raman and stimulated Raman scattering (SRS), FTIR imaging, synchrotron radiation-infrared (SR-IR) and optical-photothermal infrared (O-PTIR) microscopy with multivariate statistical analysis, we could identify marked differences in (cellular morphology and) lipid content between the two study groups. The morphological differences and potential pathological origin were also validated with immunohistochemical staining of astrocytes and microglia. These results can aid in the understanding of the functional role of reactive glial cells from molecular views and further provide new insights into the pathogenesis of AD in preclinical studies at a subcellular level.