Doxorubicin hydrochloride is a widely used anticancer agent requiring precise quantification in biological matrices for therapeutic drug monitoring and pharmacokinetics. Conventional High performance liquid chromatography technique depends on hazardous solvents and produces significant excess, posing environmental and safety concerns. The research aimed to advance and legalize a innovative, ecological reverse-phase HPLC method for quantifying doxorubicin in human plasma, allied with green analytical chemistry principles. A green HPLC technique was established employing ethanol and ammonium acetate buffer 10Mm of pH 4.5 as the mobile phase. Sample preparation employed ethanol-based protein precipitation to minimize solvent hazard. Method optimization was achieved through Box–Behnken Design, evaluating the impact of pH, rate of flow, and ethanol concentration on time of retention, area of peak, and the tailing factor. A second-order quadratic model was fitted and validated via ANOVA. Method validation followed ICH Q2(R1) and FDA guidelines. Greenness was assessed using AGREE and GAPI tools. Optimal chromatographic conditions (pH 4.5, 1.0 mL/min, 50% ethanol) yielded sharp, symmetric peaks with excellent linearity (R² > 0.998), LOD of 0.015 µg/mL, and LOQ of 0.050 µg/mL. Method demonstrated <2% RSD for precision and 98–102% recovery. Stability studies confirmed analyte robustness. AGREE scoring (0.78) and GAPI pictogram confirmed minimal environmental impact. The developed method is robust, reproducible, and environmentally sustainable, offering a reliable platform for clinical and pharmacokinetic applications of doxorubicin. © 2025 Elsevier B.V., All rights reserved.