The sweet potato is a highly important crop in terms of food security worldwide, its nutraceutical properties have increased demand, generating new market opportunities. The availability of disease-free planting material ensures success in establishing this crop, achieving higher yield and better quality. This study aimed to improve the production system of high-quality planting material in greenhouse conditions through the evaluation of three mixtures of substrates based on peat, vermicompost, rice husk, and coconut substrate in different proportions; the best treatment was subsequently evaluated in the acclimatization process of in vitro plants. The substrates were characterized physicochemically and their effect on growth parameters in sweet potato seedlings was determined. For the acclimatization process of in vitro plants, the best mixture of substrates and a humid chamber during the first eight days of growth was compared to the conventional technique. A substrate composed of peat, vermicompost, and rice husk (3:1:1) resulted in the best seedling development. The proposed in vitro plant acclimatization strategy produced seedlings with good growth, high survival rates (92%), and a good multiplication rate (3.53) compared to the conventional strategy (peat without a humid chamber). The use of an optimal substrate and the incorporation of a humid chamber during the first days of growth guaranteed adequate ranges of temperature and relative humidity that kept the vapor pressure deficit of the leaves below critical levels (< 1.2 kPa). Increased efficiency in the production of high-quality planting material with carefully controlled phytosanitary conditions can make an important contribution to improving global disease management strategies in sweet potato cultivation.