Transfer banana plantlet from in vitro to ex vitro conditions is essential prerequisite in micropropagation. In vitro grown plantlets are unable to cope with soil environment. To avoid these challenges, the plant must be acclimatized according to a protocol that takes into account the economics of the plant concerned. Banana shoot culture was established on MS medium supplemented with 5 mg/l BAP and 30 g/l sucrose. Shoot cuttings were subjected for in vitro root formation on half strength MS medium containing 1 mg/l IBA. Plantlets were acclimatized under tissue culture room conditions where they were able to repair their physiological disorders and cope ex vitro conditions. Disorder repair processes were better if plantlets were treated with chitosan. When chitosan was used, efficiency of acclimatization increased during all acclimatization periods due to the increase in plant growth parameters, water content and ROS scavenging through the increase in activities of antioxidant enzymes such as superoxide dismutase and peroxidase. To save time and costs, root formation and acclimatization were combined in one step under transparent plastic bags (relative humidity 87%) in tissue culture room (25 oC) using ex vitro- hydroponic or soil-root formation procedure. Survival frequency was 100% when root formation was induced using 1 mg/l IBA and relative humidity was gradually decreased. Plantlets were transplanted in soil without application of in vitro or ex vitro root formation procedures died in five days.