The Folin–Ciocalteu method remains the standard approach for quantifying total phenolics in plant extracts; however, matrix-specific optimization is essential for obtaining accurate results for chemically complex botanical materials. The Peruvian Amazon harbors extensive botanical biodiversity, including numerous medicinal species with uncharacterized phenolic profiles. This study developed and validated a Folin–Ciocalteu method specifically optimized for twelve ethnomedicinal plants representing eleven families from the Peruvian Amazon, following ICH Q2(R2) guidelines. Method optimization established optimal analytical conditions: 765 nm wavelength, 60 min reaction time, 14.05% sodium carbonate, and gallic acid as the reference standard. Comprehensive validation demonstrated excellent linearity (R² = 0.995–1.000), specificity confirmed through parallel standard addition curves (slope differences < 3%), precision with relative standard deviations below 2.63% for both repeatability and intermediate precision, and accuracy with recovery of 89.43 ± 2.76% meeting AOAC guidelines for complex matrices (80–120%). Robustness testing via response surface methodology confirmed method stability across variations in sodium carbonate concentration (7.50–14.05%), Folin–Ciocalteu reagent dilution (50–100%), and reaction time (30–90 min). Limits of detection and quantification were 4.43 and 13.44 μg/mL, respectively. Application to the twelve species revealed 10-fold variation in total phenolic content (24.6 ± 2.1 to 256.8 ± 4.3 mg gallic acid equivalents per gram dry extract), with Aspidosperma schultesii leaves exhibiting the highest concentration. This validated methodology provides a reliable analytical framework for the quality control and standardization of Amazonian medicinal plants, supporting bioprospecting efforts and therapeutic development.