Human periodontal ligament (PDL) is a membrane-like connective tissue interposed between the tooth root and the alveolar bone of which the main component is represented by collagen fibers. This tissue has an important role in supporting the tooth in the bone socket of the jaw and also in maintaining homeostasis of the surrounding tissues, such as alveolar bone and cementum. During the early stage of the application of orthodontic forces, different changes occur in PDL. For this reason, its characterization with conventional and non-conventional techniques can be extremely interesting. We investigated samples of PDL of orthodontic patients, aged between 13 and 21 years, treated with extraction of upper and/or lower premolars using two different vibrational techniques: Fourier Transform Infrared (FT-IR) spectroscopy and Raman microspectroscopy (m-RS). FT-IR spectra were obtained with a Perkin Elmer Spectrum One FT-IR spectrometer in a transmission geometry using KBr pellets. m-RS spectra were obtained with a He-Ne laser and a Jobin-Yvon TriAx 180 monochromator, equipped with a liquid N₂ cooled CCD and a grating of 1800 grooves/mm.FT-IR and m-RS spectra were analyzed in terms of convoluted peak functions to determine the basic vibrational modes that contribute to the FT-IR and Raman signal by using a best-fit peak fitting routine of GRAMS software based on the Levenberg-Marquardt nonlinear least square method. Lorentzian-Gaussian and Lorentzian curves were used for infrared and Raman spectra, respectively. Peaks constituting the spectrum were manually selected in order to define the starting conditions for the best-fit procedure. The best fit was then applied to determine the optimized intensity, position, and width of the peaks. The performance of the procedure was evaluated by means of the chi-2 parameter. Biochemical characterization of PDL tissues with clear evidence of contributions from collagen, lipid, and other protein was obtained. The analysis of Amide I and Amide III components was also performed giving an indication about the protein secondary structure. In addition, morphological characterization of PDL samples was carried out by using a Supra 40 Zeiss Field-emission scanning electron microscope.