Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process. Physical properties of protective solutions such as glass transition and collapse temperature, are key elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter provides a stepwise approach for developing a protective formulation for long term preservation of LAB and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried products are described.