The purpose of this study was to examine the potential application of Sorbitol, Sucrose and Gelatin in combination with glycerol in order to Theileria annulata infected cell line cryo-preservation. The efficiency of the freezing methods was compared by assessing the viability and plating efficiency of cells after resuscitation, dilution and equilibration in cell culture medium. As a result, the conventional cryo-preservative medium containing 5% PEG treated Bovine Serum and 10% glycerol successfully cryo-preserved cell as efficiently as the standard medium containing 80% FBS and 10% glycerol and was superior to all thirteen different experimental cryo-preservative media. A crucial result was obtained among the thirteen different serum-free media which showed that the 0.2M sorbitol+10% glycerol method yielded cells with viability of 54% and good plating efficiency results. It was concluded that the conventional method would be the best and ideal for the cryo-preservation and storage of Theileria annulata-infected cells for vaccine purposes to date.