Freeze-drying (or lyophilization) is widely used to preserve thermo-sensitive active ingredient such as proteins or microorganisms. Our objective was to investigate the effect of the product thermal history during the freeze-drying process on the acidification activity of lactic acid bacteria. An empirical model describing the degradation of the acidification activity of Lactobacillus bulgaricus CFL1 during the process as a function of the temperature difference between the product and the glass transition temperature (Tg) was developed. This “quality” model was associated to a heat and mass transfer model of the freeze-drying process and the losses of acidification activity during the process were simulated for various operating conditions of shelf temperature and chamber pressure. Low losses of acidification activity were predicted when the process was performed at high values of product temperature, i.e. when the time the product temperature exceeds the glass transition temperature where molecular mobility increased was minimized. Two freeze-drying cycles were carried out at low and high values of product temperatures and the experimental results of the acidification activity of bacteria were in good agreement with the simulation results. This freeze-drying model including the prediction of the degradation of lactic acid bacteria appears as an interesting tool for designing optimal processes.