The objective of this work was to assess the impact of various NaCl concentrations on Lactobacillus rhamnosus OXY viability after freeze-drying. Osmotic stress was applied during the exponential growth phase of bacterial culture. At salt concentrations between 0.2–0.5 M, a high biomass concentration and a significant increase in cell viability after lyophilisation was observed. An analysis of two-dimensional protein gels indicated the presence of shock proteins, for example, GroEL, ClpB, DnaK, TF, which provide resistance during freeze-drying and subsequent storage. On the basis of these results, it is recommended that lactic acid bacteria cultures be sub-lethally treated with 0.5 M NaCl before freeze-drying.