<p>This study aimed to validate UV-Vis spectrophotometric methods for the determination of total flavonoid content (TFC), total polyphenol content (TPC), and total antioxidant capacity (TAC) in aqueous leaf extracts of <i>Anemopsis californica</i> obtained by ultrasonic probe-assisted extraction. The methods demonstrated excellent linearity within the 0.00–0.08 mM range, yielding coefficients of determination (<i>R</i>²) of 0.9995 (TFC), 0.9984 (TPC), and 0.9972 (TAC). The established limits of quantification were exceptionally low (0.0083 mM for TFC, 0.0145 mM for TPC, and 0.0099 mM for TAC), while the accuracy was supported by high recovery rates ranging between 95.73% and 97.36%. Methodological evaluation of the extraction performance determined that a solid to liquid ratio of 0.125 g of leaf material per 10 mL of water provided the optimal analytical response. Under these standardized conditions, initial extract values reached 43.63 mg QE·g⁻¹ (TFC), 16.70 mg GAE·g⁻¹ (TPC), and 17.93 mg GAE·g⁻¹ (TAC). Furthermore, a 4-week refrigerated storage study (4 °C) revealed differential stability profiles, and TPC and TAC remained highly stable, whereas TFC underwent significant degradation, underscoring the lability of flavonoid-associated responses over time. Complementary DPPH assays confirmed relevant radical scavenging activity, which varied notably depending on the plant material source (collected versus commercially leaves). Additionally, preliminary in vitro cytocompatibility assessments showed no evidence of cytotoxicity toward human dermal fibroblasts and J774A.1 macrophages. Overall, this work provides a robust, validated analytical platform for evaluating <i>A. californica</i> aqueous extracts, establishing a reliable foundation for future detailed phytochemical profiling and mechanism-oriented biological assays.</p>