ABSTRACT The excystation of sporozoites from the oocyst of genus Eimeria is essential for conducting successful in vitro assays on the parasite. We tried to find the most efficient excystation conditions using glass beads for the in vitro excystation of E. tenella oocysts. The oocyst suspension was mixed with glass beads in a ratio of 1:1, and vortexed by various combinations of vortexing times and speeds in a Vortex Mixer. We analyzed the sporocyst-release data by regression analysis, and the peak point (duration) of excystation was predicted after differentiation of the obtained regression equation. The results indicated that the 1-mm glass beads at 2,000 rpm could release the maximum number of sporocysts in 1.64 min. Thus, excystation of E. tenella oocysts was considered to be the most effective under these conditions. Our data presented the best conditions for efficient excystation of E. tenella oocysts.