Microbial contamination is one of the most serious problems of plant tissue culture. Treating the mother plant withchemicals and nourishing them in a greenhouse instead of letting them to laboratory directly from the field hasgreater importance to plant tissue culture techniques to reduce contamination los and to increase potency level. Inthe present study, effort was made to develop an effective surface sterilization and shoot imitation protocol with anenhanced survival rate of nodal explants of vanilla by treating with various sterilant combination with differentexposure time and hormonal combinations to enhance initiation. The explants were collected from JimmaAgricultural Research Center greenhouse and used for the subsequent experiments. In all cases, the experimentswere laid out in Completely Randomized Design (CRD) with factorial treatment combinations and replicated threetimes. In this study, statistically highly significant (p<0.001) differences were recorded from the use of differentconcentrations of active chlorine in local bleach (Berekina®) and varied levels of treatment durations on asepticculture initiation. Therefore, the best result 82% rate of asepsis with survival rate of 80% was obtained from the useof 25 minutes treatment of nodal explants using a solution Berekina® containing 5% active chlorine. Regarding shootinitiation, the combined use of BA and Gibberellic Acid (GA3) revealed statistically very highly significant (p<0.001)differences, whereby 1.5 mg l-1 BA and 0.5 mg l-1 GA3 proved to be the best providing the mean length of 6.67 cm;node number 6 and mean leaf number 5 of shoot after five weeks of culture was initiated.