Fourier Transform InfraRed spectroscopy (FTIR) is an emerging technique in biology. One of the analytical problems inherent with this approach is the extreme variability typical of biological systems. Extrinsic fonts of variations are the growth media and the growth phase of the microbial culture. In order to gain knowledge on the relations between these two factors and the spectral variability, metabolomic changes in Saccharomyces cerevisiae cells during growth were analyzed with FTIR spectroscopy. Spectral variations reflected the changes occurring in all cellular compartments and components during the different growth stages and describe the metabolomic evolution of cell cultures during growth. Three different media were tested. Different mathematical and statistical treatments were used to describe and evaluate the changes occurring during growth. Variations were mainly concentrated in the exponential phase, whereas fewer changes occurred in stationary phase cells. The three different media caused different metabolomic behaviours during growth. The mathematical distance between the peak areas of amide I, amide II and of two distinct peaks in the carbohydrates region could give a synthetic description of growth and produced similar trends in all the conditions tested. The distance between the two carbohydrate peaks could be used to determine the mid exponential phase and the entrance in the stationary phase. Growth phase was shown to influence the sensitivity of a FTIR-based cell stress bioassay. The knowledge of the metabolomic variations during growth could be used in the future to optimize FTIR applications in microbiology.