<p>Naloxone hydrochloride, a life-saving opioid antagonist, plays a vital role in emergency overdose management. While numerous high-performance liquid chromatography and ultraviolet methods exist for its estimation, a critical gap remains in the use of high-performance thin-layer chromatography (HPTLC) for its quantitative analysis and degradation profiling. This study focuses on the development and validation of a robust, environmentally friendly HPTLC method for the quantification of naloxone hydrochloride, guided by the principles of quality by design (QbD). The method also encompasses the detection and characterization of degradation products under stress conditions recommended by the International Council for Harmonisation. A Box–Behnken design was applied to optimize critical chromatographic variables. Stress degradation studies were performed under all the conditions. Characterization of the degradation products was carried out using high-resolution mass spectrometry and infrared spectroscopy (IR). The method’s environmental sustainability and practical utility were assessed using Analytical GREEnness Metric, Green Analytical Procedure Index, and Blue Applicability Index assessment tools. The method exhibited excellent linearity (250–1500&#xa0;ng/band, <i>R</i>² = 0.9986), high precision, and robust recovery (98.93–100.97%). Degradation was observed under all stress conditions, with significant degradation under oxidative condition (32.93%). Mass spectrometry (MS) and infrared (IR) analysis confirmed the presence of distinct degradation products, revealing new insights into naloxone’s degradation pathways. Greenness assessments indicated the method’s strong environmental compatibility. This is the first reported QbD-based HPTLC method for naloxone hydrochloride with comprehensive degradation profiling and green chemistry evaluation. The method is simple, rapid, sustainable, and ideal for routine quality control in pharmaceutical industries.</p> Graphical abstract <p></p>