Background: Hormonal imbalances related to women's health, physical activity, and fluctuations are prevalent metabolic disorders in several nations and have significantly impacted women's health for an extended period. The application of individual or combined botanical extracts in traditional, alternative, and complementary medicine is employed to manage and alleviate these issues. Objectives: The objective of this study is to examine the suppressive properties of horehound (Marrubium vulgare L.) on pivotal hormones associated with feminine disorders. Methods: The horehound plant was exposed to ultrasonic radiation while five different solvents (methanol, ethyl acetate, n-hexane, acetone, and water) were used to extract its components. The individuals were isolated using high-performance thin-layer chromatography (HPTLC). The most powerful compounds were analyzed using a direct antioxidant assay (DPPH test) and a hormone inhibitory assay (Oestrogen, Progesterone, Estradiol, and Testosterone) on the HPTLC plate. The compounds that had a significant effect were then identified using LC-ESI/MSMS. Results: The antioxidant properties of the extracts and hormone inhibitors were evaluated, and the substances were separated from the HPTLC plate and analyzed using mass spectrometry. The results showed strong antioxidant capabilities, with an IC50 range of 8.24 - 12.42 µg/mL. Moreover, the plant extract showed beneficial effects on hormones associated with female health issues. Conclusions: The extract was subjected to chemical and molecular analysis using the HPTLC technique, followed by LC-ESI/MSMS. The study revealed the presence of vulgarole, marrubiin, and marrubenol chemicals.