The aquaporins 3 (AQP3) are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across skin cell membranes. The recent findings revealed that AQP3 are involved in the progression of skin disorders, such as atopic dermatitis, psoriasis, eczema, vitiligo, and xerosis. Research of novel combination of plant molecules that could increase the expression of this protein for skin hydration is currently ongoing. Through DiffDock computational modelling to predict affinity for AQP3 and the biological activity, Aloe vera extract and trimethylglycine were chosen. Thus, our science work was focused on the development of a novel combination based on Aloe vera extract and trimethylglycine and evaluation of targeted AQP3 regulation in skin keratinocytes in the presence of this combination. Firstly, the cytotoxicity assay of selected substances was performed with MTT indicator on HaCaT cells. Secondly, the substances’ ability to increase amount of AQP3 was evaluated in the keratinocytes’ cell culture with ELISA immunoassay. According to the results obtained, the novel combination based on Aloe vera extract and trimethylglycine in a mass ratio of 1:1 had a good cytotoxicity profile, with an EC70 value of 11.95%. Moreover, it was shown that the combination had a clear synergetic activity and significantly increased amount of epidermal AQP3 up to 219%, compared to that of negative control (p<0.001). Thus, the novel combination of plant molecules has a promising potential for the development of dermatological drugs and the treatment of skin disorders related to the low skin hydration.