The global outbreak of the novel coronavirus (COVID-19) has highlighted the urgent need for innovative therapeutic solutions. Remdesivir (REM) was the first drug granted approval by the US FDA for treating hospitalized COVID-19 patients. A selective and sensitive derivative spectrofluorimetric method has been developed and validated for the determination of Remdesivir (REM) in presence of its Alkaline-induced degradation product (AKDP), which is also known to be its metabolite (GS-441524). The method utilized the intrinsic fluorescence properties of REM, achieving a linear response within the range of 3.0–120.0 ng/mL at 428.3 nm using first-order derivative. Methodological parameters were optimized to ensure high sensitivity, with detection and quantification limits of 1.12 and 3.67 ng/mL, respectively. This approach successfully quantified REM in pure form, intravenous infusions, and spiked human plasma. Recovery rates in plasma were satisfactory at 97.64 ± 1.87, confirming the method’s suitability for therapeutic drug monitoring (TDM) in COVID-19 patients. Additionally, the environmental sustainability of the method was evaluated using GAPI, AGREE, and RGB12 metrics, underscoring its green and eco-friendly characteristics.