Abstract: The research focused on the preparation of Directed-Vat-Set( DVS) yoghurt starter cultures using Propionibacterium freudenreichii through Vacuum Freezing Drying technique and the optimization of its condition through single factor and response surface assays. The results showed that skimmed milk,sucrose and sodium glutamate were more effective than gelatin,glycerol,sorbitol through determination of acidity and viable bacteria count in single factor assay. The optimal condition was as follows: skimmed milk 11. 82%,sucrose 7. 03%,sodium glutamate 2. 98%. At the best condition,yoghurt fermented by this DVS had a acidity of 69 ± 0. 2425,which was approaching predicted value of 71. 0683.