Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the “microcollapse” temperature ( Tμc, beginning of a local loss of structure) and the “collapse” temperature ( Tc, beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 °C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of “robustness” to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 °C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 °C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temperature Tc was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.