This paper reports an experimental investigation based on vibrational spectroscopy of biological materials by a new custom-made device allowing simultaneously applying tensile load with changes within a controlled chemical environment. The response of the molecular structures of Temporomandibular Joint (TMJ) discs under simulated daily mechanical stimulations was characterized by stress–strain analysis and Raman spectra. The results show that changes in the biochemical environment around tissue (associated to common disorders) led to significant modifications to its mechanical properties. The molecular response to stress was then fully characterized by a combination of molecular mapping followed by statistical analysis via Principal Component Analysis resulting in the identification of the phenylalanine-aromatic amino acids as a significant mechanical-chemical effector of TMJ discs. The coupled tensile machine and vibrational spectroscopy approach enabling in-situ molecular studies proves to be a powerful technique for biological material characterization and tissue engineering.