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This study aimed to optimize ethanol-based extraction of Justicia gendarussa leaves and characterize the optimized extract through UHPLC-HRMS metabolite profiling, with application to cultivated plant materials. Methods: A two-level half-fractional factorial design was used to assess the effects of five extraction factors\u2014liquid-to-solid ratio, temperature, ethanol concentration, extraction time, and pH\u2014on three response variables: total flavonoid content (TFC), DPPH radical-scavenging activity, and ferric reducing antioxidant power (FRAP). The optimized condition was validated experimentally, characterized by UHPLC-Q Exactive Plus Orbitrap HRMS with data-dependent MS\/MS, and applied to cultivated leaf and stem materials under six fertilization treatments, analyzed using two-way ANOVA (organ \u00d7 treatment). Results: The liquid-to-solid ratio \u00d7 temperature interaction was the dominant determinant of TFC, and the liquid-to-solid ratio alone dominated both DPPH and FRAP. Desirability-based optimization identified a condition of 100 mL g\u22121 liquid-to-solid ratio, 30 \u00b0C, 20% ethanol, 30 min, and pH 2. Confirmatory extraction resulted in 16.43 \u00b1 0.30 mg QE g\u22121 DW TFC, 12.30 \u00b1 0.02 \u00b5mol TE g\u22121 DW DPPH, and 190.56 \u00b1 2.58 \u00b5mol TE g\u22121 DW FRAP, in close agreement with model predictions (0.10\u20131.54% error). UHPLC-HRMS\/MS detected 52 putatively annotated metabolites, including a candidate gendarusin A\/B (apigenin di-C-arabinoside)-type C-glycosylflavone. Leaves showed significantly higher TFC, CUPRAC, and FRAP than stems regardless of fertilization treatment (organ effect p &lt; 0.0001), whereas the leaf\u2013stem difference in DPPH capacity varied significantly with treatment (interaction p = 0.0010). Conclusions: These findings provide a validated extraction condition for the measured responses and support further targeted metabolite evaluation of J. gendarussa.<\/jats:p>","DOI":"10.3390\/ph19091466","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T09:06:37Z","timestamp":1789549597000},"page":"1466","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fractional Factorial Design-Guided Optimization of Flavonoid Content and Antioxidant Capacity in Justicia gendarussa Leaf Extracts and UHPLC-HRMS-Based Metabolite Characterization"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7047-5093","authenticated-orcid":false,"given":"Waras","family":"Nurcholis","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Faculty of Mathematics and Natural Sciences, IPB University, Bogor 16680, Indonesia"},{"name":"Tropical Biopharmaca 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