Istradefylline, a selective adenosine A2A receptor antagonist used as an add-on therapy to levodopa therapy for Parkinson’s disease, requires reliable impurity profiling to ensure pharmaceutical quality and safety. In this study, a novel, stability-indicating, Quality by Design (QbD)-based and eco-friendly RP-HPLC method was developed and validated for the determination of Istradefylline and its related impurities in tablet dosage forms. Method robustness was systematically evaluated using a three-level, two-factor factorial design. Chromatographic separation was achieved on an XBridge C18 column (250 × 4.6 mm, 5 µm) using 0.01M ammonium formate buffer (pH 4.0) and acetonitrile at a flow rate of 0.8mL min⁻¹, with detection at 280 and 360nm. The method demonstrated excellent specificity, effectively separating Istradefylline from its impurities (IST-4, IST-5, IST-CIS, IST-10, and IST-40) without interference from excipients and degradation products. Linearity was established over the concentration range of 0.200–4.000µgmL⁻¹ (R² > 0.999), while accuracy, precision, and sensitivity met ICH acceptance criteria. Forced degradation studies confirmed the stability-indicating capability of the method. The method also complies with Green Analytical Chemistry principles through reduced solvent consumption and efficient chromatographic performance. Validation results demonstrated that the method is robust, reliable, and suitable for routine quality control, impurity profiling, and stability assessment of Istradefylline tablet formulations.