Fosaprepitant dimeglumine, an injectable phosphorylated prodrug of aprepitant, has been approved for preventing chemotherapy-induced nausea and vomiting. A novel stability-indicating HPLC method was designed and validated to determine process- and degradation-related impurities of fosaprepitant dimeglumine in an injection formulation. Chromatographic separation was done on a NanoChrom C18 (250 mm×4.6 mm, 5µm) column at a column oven temperature of 35°C. Mobile phase A had 0.5 M ammonium dihydrogen phosphate solution (pH fixed to 2.2 with orthophosphoric acid) and acetonitrile at 80:20 ratio and mobile phase B had methanol and acetonitrile at 70:30 ratio. The formulations underwent forced degradation conditions, like acidic, basic, thermal oxidation and photolytic conditions. The designed HPLC approach was validated per International Conference of Harmonization (ICH) guidelines, including limit of detection (LOD), specificity, limit of quantitation (LOQ), accuracy, linearity, precision and robustness. The results showed that this method is specific, sensitive, precise, accurate and robust.