High quality probiotic powder can lay the foundation for the commercial production of functional dairy products. The freeze-drying method was used for the preservation of microorganisms, having a deleterious effect on the microorganisms viability. In order to reduce the damage to probiotics and to improve the survival rate of probiotics during freeze-drying, the Response Surface Methodology (RSM) was adopted in this research to optimize lyoprotectant composed of amino acids (glycine, arginine) and salts (NaHCO3 and ascorbic acid). Probiotic used was