The chromatographic analysis of pharmaceutical impurities and degradation products is essential for ensuring drug quality and stability. In this study, a selective, sensitive, and straightforward high-performance thin-layer chromatography (HPTLC) method was developed for the simultaneous determination of Remdesivir (REM) in presence of its alkaline-induced degradation product in both bulk and pharmaceutical formulations. Separation was achieved on HPTLC silica gel 60 F254 plates using a mobile phase consisting of ethyl acetate, methanol, and ammonia (33%) in the ratio of 8:2:0.2 (v/v/v). Densitometric detection was performed at 232 nm, and polynomial regression models were used to construct calibration curves. Linearity was observed over the concentration range of 0.2–1.1 µg/band for REM. Various method parameters—including mobile phase composition and detection wavelength—were systematically optimized to ensure high resolution, accuracy, and sensitivity. System suitability parameters were also assessed to confirm the method’s performance. The developed method was validated according to International Council for Harmonisation (ICH) guidelines and was successfully applied for the quantitative determination of REM in pharmaceutical dosage forms, without interference from formulation excipients. The results were statistically compared with those obtained from a reference HPLC method showing no significant differences. Additionally, the method’s environmental impact was evaluated through a greenness assessment, supporting its suitability for routine quality control applications.