Abstract: In view of the absence of analytical methods for related substances of the investigational new drug Namodenoson, this study established and validated a stable and reliable high-performance liquid chromatography (HPLC) method for the first time. The analysis was performed on a Luna C₁₈ column (4.6 mm × 250 mm, 5 μm), using a mobile phase consisting of 2 mmol/L potassium dihydrogen phosphate aqueous solution (adjusted to pH 2.5 with phosphoric acid)–acetonitrile (90: 10, v/v) as mobile phase A and pure acetonitrile as mobile phase B, with gradient elution. The detection wavelength was set at 275 nm, the column temperature at 35 °C, and the flow rate at 1.0 mL·min⁻¹. To address the difficulty in obtaining reference standards for process-related and degradation impurities of the new drug, an innovative approach was employed in which ultraviolet light–induced degradation solution and alkaline degradation solution were mixed in equal volumes to construct a system suitability solution. This approach effectively covered multiple potential degradation products and achieved complete separation of difficult-to-resolve peak pairs. Method validation results showed that the resolution between the main peak and adjacent impurity peaks reached 4.28, and the minimum resolution between impurity peaks was 1.69. Namodenoson exhibited good linearity within the range of 0.10–10.17 μg/mL (r = 0.9989), with the limit of detection (LOD) and limit of quantification (LOQ) determined to be 0.013 and 0.05 μg/mL, respectively. Forced degradation studies demonstrated that the method had strong specificity, with mass balance ranging from 96.06% to 102.17%. The method established in this study is stable, reliable, and robust, and can provide critical analytical support for the establishment of quality standards, stability studies, and process optimization of Namodenoson.