The medicinal plant Hypericum perforatum (St. John's Wort) is of great pharmaceutical importance due to its bioactive compounds, such as hypericin and hyperforin, which exhibit antidepressant, anti-inflammatory, and neuroprotective activities (Suryawanshi et al., 2024). However, the natural production of these secondary metabolites is limited and influenced by environmental and genetic factors. In vitro culture techniques, particularly callus culture, offer an effective approach to enhance and regulate their production. Abiotic stresses, such as salinity, can stimulate biosynthetic pathways, increasing the accumulation of phenolic compounds, flavonoids, and antioxidant enzyme activities, which contribute to stress tolerance and cellular defense. Considering this, the present study investigated the effect of different NaCl concentrations on secondary metabolite production and antioxidant activity in H. perforatum callus under in vitro conditions. Seeds were surface-sterilized and germinated, and leaf explants were cultured on MS medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L BAP for callus induction. After callus formation and one subculture, calli were transferred to media containing 0, 50, 100, or 150 mM NaCl. Total phenolic and flavonoid contents were measured using standard spectrophotometric methods, and antioxidant capacity was assessed by a DPPH assay. The results showed that salinity elicitation significantly enhanced secondary metabolite accumulation and antioxidant activity. The highest production of phenolics, flavonoids, and antioxidant activity was observed at 150 mM NaCl, while the lowest values were recorded in the control (0 mM). These findings indicate that elicitors can effectively simulate environmental stress and stimulate biosynthetic pathways, enhancing the production of pharmacologically valuable compounds in H. perforatum.