The tumor microenvironment, or stroma, is chemically and morphologically modified during carcinoma progression. The predominant cell type in the stroma, the fibroblast, maintains collagen properties in normal tissue and often transformed during tumor progression. Biochemical changes within fibroblasts upon initial cancer activation, however, are relatively poorly defined. Here, we hypothesized that Fourier transform infrared (FT-IR) spectroscopic imaging could potentially be employed to examine these early transformations. Further, we employ attenuated total reflectance (ATR) microscopy to characterize subcellular spectra and their changes upon transformation. We characterized fibroblast transitions upon stimulation with both a molecular agent and a carcinoma-mimicking cellular co-culture system. Changes were predominantly observed in the 1080 cm−1 and 1224 cm−1 peak absorbance, commonly associated with nucleic acids, as well as in the band at 2930 cm−1 associated with the C–H stretching of proteins in the cytoplasmic compartment. In conclusion, biochemical changes in cancer-associated fibroblasts that express α-SMA are dominated by the cytoplasm, rather than the nucleus. This ensures that spectral changes are not associated with proliferation or cell cycle processes of the cells and the cells are undergoing a true phenotypic change denoted by protein modifications in the cell body.