Alteration of cell membrane permeability is speculated to be one of the damage mechanisms to Lactic Acid Bacteria during freeze-drying. It has been reported that the H+ and Ca2+ alteration may be accurately detected by using fluorescent probe. The Lactobacillus bulgaricus permeability is studied by using fluorescent probe BCECF-AM and Fluo3-AM. The membrane permeability has a significant increase which is anti-related to the loss of vitality. These results suggest that alteration in cell membrane permeability contributes to the physical damage, it may be one of the reasons that lead to the cell death and loss of activity.