This study delves into the antioxidant potential of Justicia gendarussa, commonly known as gendarussa, and its response to varying doses of nitrogen fertilizer. Gendarussa exhibits the potential for antioxidant activity. The diverse ecological conditions in which it thrives may influence its biological activity and lead to inconsistent production of secondary metabolites. Nitrogen, a pivotal factor in secondary metabolite production in plants, has become a focal point of this research. This research aims to determine the optimal nitrogen fertilizer dose on gendarussa antioxidant capacity and metabolites using a metabolomics approach. Employing a randomized block design for cultivation, the investigation revealed that a maximum harvest weight of 10.9 g/aerial parts of the plant was achieved with 270 kg/ha of nitrogen fertilizer. This study explored the DPPH, ABTS, FRAP, and CUPRAC assays of antioxidant effect, and found insignificant differences between the various nitrogen treatments. UHPLCQ-Orbitrap HRMS was employed to identify 30 metabolites in positive and 18 in negative ionization modes. Gendarusin A, a major metabolite in gendarussa, is identified in both positive and negative ionization. PCA and heatmap analysis successfully categorized these metabolites in the aerial parts of gendarussa at different nitrogen fertilizer dosages. Based on the metabolomics approach, variations in nitrogen fertilizer made metabolites at doses of 90 kg/ha had higher relative concentrations of metabolites compared to doses of 180 kg/ha and 270 kg/ha. So, 90 kg/ha are the optimal nitrogen fertilizer dose for cultivation and utilization strategies.