Salvia plebeia R. Brown is a medicinal plant containing various bioactive compounds, including flavonoids, and has been reported to exhibit diverse pharmacological effects such as anti-inflammatory, antioxidant, and hepatoprotective activities. However, previous studies on S. plebeia have mainly focused on phytochemical identification and pharmacological evaluation, while biotechnological approaches aimed at enhancing the productivity of major bioactive compounds—particularly metabolic regulation and content improvement through elicitor treatment in in vitro culture systems—remain largely unexplored. In this study, we investigated the effects of elicitor treatments on the accumulation of homoplantaginin, a major bioactive compound in the leaves of in vitro–cultured S. plebeia, as well as the associated changes in anti-inflammatory activity. In vitro–grown plantlets were treated with yeast extract at concentrations of 1, 2, and 5 mg/L and polyethylene glycol (PEG) at concentrations of 1, 2, and 5% (w/v), respectively. Homoplantaginin content was quantitatively analyzed using high-performance liquid chromatography (HPLC), and anti-inflammatory activity was evaluated by measuring nitric oxide (NO) production inhibition in RAW 264.7 macrophage cells. As a result, the homoplantaginin content was significantly increased in the treatment with 2 mg/L yeast extract compared to the control, while the highest NO inhibition activity was observed in the 5% PEG treatment. These findings suggest that elicitor treatment can effectively enhance the production of bioactive compounds and anti-inflammatory activity in in vitro–cultured S. plebeian. Furthermore, this study provides fundamental data supporting the potential industrial application of S. plebeia through further elucidation of metabolic pathways and optimization of culture conditions.