Kefir is a probiotic food produced by the fermentation of different substrates by Kefir grains, composed of bacteria and yeasts. Kefir presents greater microbiological diversity in comparison to other probiotics. Therefore, to increase stability and maintaining cell viability, lyophilization is presented as a good alternative because is a process obtained by sublimation under vacuum minimizing various degradation reactions occurring during drying. However, the frozen that precedes it is critical, elucidating the importance of cryoprotectants to avoid lioinjury. The objective of this study was to evaluate the effects of maltodextrin and sucrose on the cryoprotection and cell viability of microorganism of freeze-dried kefir. The fermentation with kefir grains was carried out with pasteurized whole milk in static conditions for 24h at 25ºC. The complete factorial design 22 with a central point (totaling 7 trials) was used to evaluate the effect of the independent variables: concentration of maltodextrin and sucrose on the dependent variable: total counts of bacteria (MRS and M17 media) and yeasts (YGC media) expressed in log CFU.g-1, as well as identification of genera in the respective culture media according to the morphological and physiological characteristics. Kefir was evaluated before and after lyophilization by microbiological characteristics. The results evidenced the greater influence of sucrose on the preservation of microorganisms. The best drying condition (sucrose, 100 g.L-1) provided bacterial survival of 97.71% on MRS agar and 96.68% on M17 agar and yeasts survival of 89.63% on YGC agar. The five different genera Enterococcus spp., Acetobacter spp., Bifidobacterium spp., Lactobacillus spp., and Candida spp. was identified in descending order. Thus, it was demonstrated a satisfactory maintenance of cellular viability by the technology employed and because of the process of addition of cryoprotectant to Kefir and the production of its powder version this study is being evaluated to obtain a patent register.