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This work evaluated the effect of in vitro drought stress on morphological characteristics, phenolic compounds, flavonols, saponin content, and antioxidant activity in plantlets of Agave salmiana. Drought stress was induced with polyethylene glycol (PEG) at 0, 10, 20 and 30% w/v in Murashige and Skoog solid medium. The determination of specific flavonols and saponins was achieved via HPLC-DAD and HPLC-ELSD, respectively. Compared with the control, plants grown in 30% PEG showed a change in the width of the leaves and a different color, showing less clarity and more darkening (L = 21.18, b = 14.27) and also had the lowest flavonol content, but the highest total saponin content (tigogenin glycoside, 163 mg of protodioscin equivalents/g dw) and the highest antioxidant activity. Total phenolic compounds did not significantly differ between treatments. Agave salmiana plants cultured in vitro increased their …