A combination of spontaneous Raman, stimulated Raman, and photothermal expansion (AFM-IR) spectromicroscopy is reported for probing the impact of different radiation doses (2–10 Gy) on U87 glioma cells ex vivo. Most significant are alterations in spectral profiles caused by radiation-induced changes, while keeping the cell fixation delay constant at 24 h. The changes in delay of the fixation ranging up to 5 d at a dose of 2 Gy were also investigated for probing cellular recovery processes of exposed cells. Both, the Raman-based and AFM-IR spectral analyses identified statistically significant spectral changes and radiation-induced alterations in cellular proteins, nucleic acids, and lipids. Specifically, these label-free approaches revealed a 3-fold and 2-fold decrease in nucleic acid and lipid content, respectively, for cells treated with 10 Gy compared to untreated control samples. This study unravels the potential of a combination of Raman-based approaches and AFM-IR that is of use for therapeutics and offers a novel way to monitor and localize radiotherapy-induced changes in tumor cells.