<p>This study proposes a sensitive and green spectrofluorimetric method for assessing brucine in solid dispersion adsorbate formulations and spiked plasma samples by combining quality by design principles with white analytical chemistry concepts. This method attains nanogram-level sensitivity by employing fluorescein as a biosensing probe through photoinduced electron transfer. The targeted formulation showed improved solubility and dissolution rates compared with those of pure brucine. In-vivo pharmacokinetic studies revealed enhanced oral bioavailability with the BRU-SDA formulation achieving a significantly higher plasma concentration than the values obtained for the physical mixture. The proposed spectrofluorimetric technique was validated and showed good linearity, accuracy, and precision across a broad range of concentrations at nanoscale levels. The Lowest Limit of Detection (LOD) and Lowest Limit of Quantitation (LOQ) were found to be 10 and 20 ng•mL^-1&#xa0;respectively, with recovery rates of 90–95% from biological samples. The method’s overall performance was evaluated using the RGB12 principles, achieving exceptional scores of 95 for redness (analytical quality), 76 for greenness (environmental sustainability), and 100 for blueness (practical utility), resulting in an outstanding whiteness score of 90.33 out of 100. This comprehensive assessment demonstrates that the proposed method outperforms current chromatographic methods in terms of analytical performance, environmental sustainability, and practical usability. While maintaining equivalent sensitivity, this spectrofluorimetric technique proved to be more time-efficient, user-friendly, and less expensive than the published techniques. These benefits position the proposed method as a viable alternative for standard analysis in pharmaceutical quality control and research environments, representing a significant step towards more sustainable and effective analytical techniques in the pharmaceutical industry.</p>