The purpose of this study was to investigate how the properties of Mexican kefir grains (MKG) are affected by the operating parameters used in the freeze-drying process. The factors investigated were the freezing time (3−9 h), freezing temperature (−20 to −80 °C), pressure (0.2−0.8 mbar), and lyophilization time (5−20 h). The maximum range of change and one-way analysis of variance showed that lyophilization time and freezing time significant affects (p < 0.05) the response variables, residual moisture content and water activity, and pressure had a significant effect on the color difference and survival rate of probiotic microorganisms. The best drying conditions were a freezing time of 3 h, a freezing temperature of −20 °C, a pressure of 0.6 mbar, and a lyophilization time of 15 h. Under these conditions, we obtained a product with residual moisture content below 6%, water activity below 0.2, and survival rates above 8.5 log cfu per gram of lactic acid bacteria and above 8.6 log for yeast.