This study aimed to establish and validate a high-performance liquid chromatography (HPLC) method for the determination of related substances in namodenoson. A system suitability solution was prepared by mixing equal volumes of the UV-degraded solution and the alkali-degraded solution, which could cover the potential major related substances. Separation was performed on a Luna C18 column (250 mm × 4.6 mm, 5 μm) using gradient elution with mobile phase A (a mixture of 2 mmol/L potassium dihydrogen phosphate solution adjusted to pH 2.5 with phosphoric acid and acetonitrile, 90∶10) and mobile phase B (acetonitrile). The detection wavelength was set at 275 nm, and complete separation of all related substance peaks was obtained. Method validation results demonstrated that the resolution between namodenoson and its related substances, as well as that among all related substances, met the requirements. Namodenoson exhibited good linearity over the range of 0.1–10 μg/mL (r = 0.9989), with a limit of detection (LOD) of 0.01 μg/mL and a limit of quantitation (LOQ) of 0.05 μg/mL. Analysis of forced degradation samples further demonstrated the excellent specificity of the method, with satisfactory mass balance values (96.1%–99.1%). The method established is stable, reliable, and robust, and can serve as a reference for the establishment of quality standards, stability studies, and manufacturing process optimization of namodenoson.