In recent years, the laser-based ablation of damaged or undesired tooth material has emerged as a highly promising technique for improving dental cavity preparation. While X-ray diffraction and X-ray photoelectron spectroscopy are generally used to characterize the constituents of ablated surfaces, Fourier Transform Infrared (FT-IR) can also be employed for monitoring the changes induced by the femtosecond laser ablation process. In the present study, FT-IR spectroscopy has been adopted to characterize the changes induced in extracted human teeth. The laser ablation was performed in ambient air by using a femtosecond laser source at different fluences in the range of 0.7–1.5 J/cm² to produce regular lines on various samples. Micro-ATR spectroscopy was employed to examine the laser-processed tooth disks. The spectra acquired from different samples reveal the contributions of the various dental components and provide insight into the effect of laser processing under different conditions.