Production of plants under artificial light conditions is an innovative and smart concept to grow food year-round in any location. However, pre-basic seed potato production in the greenhouse from LED pre-treated seedlings under an aeroponic system is a new and creative idea. Therefore, the objective of the study was to optimize the effect of LED pre-treatment and determine the best LED spectral composition on growth performance and tuberization of potato plants. Potato variety ‘Golden King’ was treated under 9 LED light spectra for 30 days—L1 (natural light), L2, (R:B), L3 (R:B:G), L4 (R:B:FR), L5 (R:B:G:FR), L6 (R:B:G:FR:UV), L7 (R:B:FR:UV), L8 (R:B:W:FR), and L9 (R:B:W:FR:UV) under 300 µmol m−2 s−1 photosynthetic photon flux density (PPFD), 23/15 °C (day/night) temperature, and 70% relative humidity. The study revealed that growth characteristics and tuber number for plants were increased most by the light spectrum L4 (R:B:FR). Furthermore, photosynthetic pigments increased in L4, L7, and L8, while TSC and sucrose accumulated more in L1 treatment. In contrast, higher seed tuber fresh weight was recorded in L8, L9, L4, and L7. Overall, it can be concluded that potato seedlings pre-treated with the L4 (R:B:FR) LED spectral composition performed best for growth, establishment, and tuberization.