This article presents a new acclimatization method, ex vitro acclimatization in float hydroculture. This protocol ensures the ex vitro acclimatization of the plantlets obtained in vitro in the rooting stage or the ex vitro rooting and acclimatization of the shoots obtained in the multiplication stage. Our hydroculture method is different from the techniques of flotation hydroculture because no fertilizers or plant growth regulators or other biostimulators are used and water oxygenation by bubbling is not provided. Ex vitro rooting and acclimatization in one stage without an in vitro rooting stage in floating cell trays was successfully carried out in Rubus fruticosus and Rosa hybrida cultivars, whereas ex vitro acclimatization of in vitro-rooted plantlets was successfully carried out in Rubus idaeus, Ribes nigrum, Prunus cerasus × P. canescens, Lycium barbarum (Goji berry), Amelanchier canadensis, Drosera rotundifolia, Drosera capillaris, and Nephrolepis sp. In another set of experiments, the floating cell trays were replaced with layers of floating perlite and the latter method was also tested for non-rooted shoots obtained in vitro in the multiplication stage or rooted plantlets obtained in the rooting stage. Direct ex vitro rooting in floating perlite was successful in ‘Tayberry’ (Rubus fruticosus × Rubus idaeus) (78.12% rooting); Amelanchier canadensis, Rubus fruticosus ‘Chester’, Rubus idaeus ‘Erntesegen’, Vaccinium macrocarpon, and Vaccinium corymbosum with rooting percentages above 80%; and Rubus idaeus ‘Willamette’ and Rosa hybrida ‘Cristiana’ with rooting percentages above 80%.