In strawberry micropropagation, several challenges must be overcome to obtain quality plants and achieve high survival rate to ex vitro acclimatization. In this study, therefore, a set of protocols were evaluated to optimize explant (meristem) disinfection, in vitro growth (multiplication and rooting), and ex vitro acclimatization of strawberry. The results showed that explants treated with 1.0% NaClO for 5 min had a lower percentage of contamination, and achieved a higher percentage of viability, height, and number of leaves. In vitro growth was favored by the use of 1 mg L−1 zeatin, since it allowed greater seedling growth (number of shoots, seedling height, number of leaves, number of roots and root length), and a higher pre-acclimation rate (100%). In the acclimatization phase, plants grown in a substrate composed of compost + peat combined with 4 g of humic acid achieved better response in morphological and physiological variables. In fact, the results of this study could be used to cultivate strawberry plants of the ‘Aroma’ variety with high commercial quality.