Aims: The aim of the present work was to evaluate the effect of various cryoprotective agents on the survival of freezedried probiotic Lactobacillus rhamnosus. Methodology and results: Investigation was done on the viability and stability of probiotics L. rhamnosus. The effect of different cryoprotective agents (namely, sodium chloride, sucrose, dextran, sorbitol, monosodium glutamate, glycerol, skim milk and skim milk with malt extract) with modified De-Man Rogosa Sharpe (MMRS) medium were examined. Commercial De-Man Rogosa Sharpe (MRS) medium was proved to be more expensive than the modified MRS medium with relatively low yield of probiotics L. rhamnosus. Significantly high viable counts were achieved with monosodium glutamate, skim milk and skim milk with malt extract, with optimum concentration at 0.3% w/v. There was a reduction in cell viability at concentration above 0.5% w/v, which could be attributed to cell shrinkage associated with osmotic pressure changes. The cells were found to be stable at room temperature (28 °C) for eight weeks. A significant growth of probiotics was produced from skim milk. Conclusions: Modified MRS medium with skim milk is suggested for the remarkable growth and yield of probiotic lactobacilli.