For the first time, an attempt was made to measure the stress level of professional volleyball players using FTIR and Raman spectroscopies. Stress is an inevitable factor accompanying every professional athlete at various stages of sports competition. A group of professional female volleyball players being at the preparatory stage for the sports season were involved in this research. The correlation between laboratory tests cortisol concentration and the FTIR absorbance value as well as Raman intensity corresponding to the vibrations of the lipid functional groups was shown. The degree of this revealed correlation was determined by the Pearson test. It was shown that correlation was best reflected by the absorbance value of FTIR spectra for the 1240 cm−1 and 2930 cm−1 wavenumbers. Moreover, the error level for the proposed spectral analysis is less than 25%. This interdisciplinary research showed for the first time the potential use of the FTIR spectroscopy method in a stress level measure.