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Polyploidization in plants, which involves doubling or further multiplying of genome, has the potential to improve the constituents that make medicinal plants, like <i>Ajuga reptans</i>, attractive to the pharmaceutical, cosmetics, and food production industries; botanical pesticide effects could also be derived. The aim of this study was to determine how artificial polyploidization in <i>A. reptans</i> plants affected the composition and quantity of biologically active substances from the glycoside and phytoecdysone families. Diploids and artificial tetraploids of <i>A. reptans</i> were analyzed. Changes in the contents of <i>trans</i>-teupolioside, <i>trans</i>-verbascoside, and 20-hydroxyecdysone were evident in the aboveground parts of the cultivated plants (e.g., leaves and flowers). The tetraploid lines of <i>Ajuga</i> plants displayed variability in, and increased levels of, <i>trans</i>-teupolioside and <i>trans</i>-verbascoside content. The 20-hydroxecdysone content was slightly higher in tetraploids. These findings indicated that <i>Ajuga</i> tetraploids could be used in breeding programs to enhance the yield of substances with potential medicinal and industrial applications.