Background: Krymsk® 5 (VSL-2) is a semi-dwarf cherry rootstock often used to produce drought-resistant varieties with high-quality fruits. The present study aims to optimize the in vitro micropropagation protocol for rootstock. Methods: At the propagation and rooting stages, growth regulators such as 24-epibrassinolide (″Epin-extra″), kinetin, 6-benzylaminopurine and indolyl-3-butyric acid were used in various concentrations. Morphological changes, pigment content, stomatal density and size and stomatal slit size for evaluation of drought resistant rootstock were quantified. Result: Our study showed the efficiency of using preparation ″Epin - extra″(1.0 ml/L) at the initial growth stage to stimulate regenerative activity, cytokinin kinetin, (0.5 mg/L) at the stage of reproduction and auxin IBA (0.5 mg/L) at the rooting stage. Application of 0.5 mg/L 6-BAP with Epin proved to be ineffective due to shoot vitrification. For the first time, the resistance of VSL-2 cherry stock to the lack of moisture was assessed using polyethylene glycol 6000 under in vitro conditions and resistance to osmotic stress in the studied samples was confirmed. The obtained results will hopefully allow scaling up the reproduction process of the VSL-2 rootstock in vitro.