The awareness regarding use of probiotics for health benefits has created a huge market worldwide. The use of probiotic cultures and its success in commercial scale is relying on the techniques employed to ensure that the potential cultures with desired functional activity are stable and viable for longer period. The present study was undertaken to statistically evaluate the effect of lyoprotectant in the suspension media containing sucrose and reconstituted skim milk on the viability of Lactobacillus acidophilus strains. The central composite rotatable design of response surface methodology was used in evaluating the survival rate and viability even after 24 months' storage of three different lyophilized strains of L. acidophilus. We also determined the exclusive use of lyoprotectant on the survival of the lyophilized strains throughout the study period without altering any of the culture conditions. The most conspicuous result to emerge from the data is that the suspension medium composed of minimal amount of sucrose (~ 1.2%) in the reconstituted skim milk (6%) was evident in maintaining a higher survival rate of lyophilized culture during 2-year storage. From this investigation, the optimized suspension media used to obtain the lyophilized concentrate of L. acidophilus could be recommended in designing dietary supplement with minimal modification for industrial application. This might reduce the manufacturing cost as well as storage loss occurring both in pharma and food sector.