Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits. However, it is sensitive to freeze-drying and storage in the dried state, thus limiting its commercial exploitation. Our objective was to identify markers of cell resistance by applying multiscale characterization to L. salivarius CECT5713 cell populations exhibiting different resistance to freeze-dried storage. Cells were produced under two different sets of production conditions differing in the culture parameters (temperature, neutralizing solution, and harvesting time) and the protective formulation composition. The culturability, membrane integrity, and cell biochemical composition assessed by Fourier transform infrared (FTIR) micro-spectroscopy were evaluated after freezing, freeze-drying, and subsequent storage at 37 °C. Membrane properties (fatty acid composition, membrane fluidity, and phospholipid organization), as well as matrix physical properties (glass transition temperature and water activity), were determined. The most resistant cells to freeze-dried storage exhibited the highest cyclic fatty acid content and the most rigid membrane. Freeze-drying and storage induced damage to membrane integrity, proteins, nucleic acids, and constituents of the peptidoglycan cell wall. From the FTIR spectra analysis, we propose the minimization of the variations of the 1058 and 1714 cm-1 vibration bands (that arise mainly from symmetric CO -C stretching and C=O stretching, respectively) induced by the freezedrying process as a marker of storage stability. We confirmed that a matrix with a glass transition temperature at least 50 °C higher than the storage temperature is crucial for L. salivarius CECT5713 storage stability. In addition, this work explored promising FTIR methods for a better understanding of the protection mechanisms involved. Highlights • Increasing the cyclic fatty acid content and membrane rigidity correlated with improved freeze-drying and storage resistance • Two FTIR bands (1058 and 1714 cm-1) associated with other cell components than membrane lipids could be considered preservation markers • A glassy matrix with a glass transition temperature 50 °C higher than the storage temperature ensured the highest storage stability