A fast, sensitive and selective RP-HPLC method was developed for extensive investigation of nitroxinil’s stability. The stability of the studied drug was tested under different stress conditions, namely hydrolytic, oxidative, photolytic and thermal. Separation of nitroxinil and its degradation products was achieved in less than 5 min using Venusil XBP C18 (150 × 2.1 mm id, 5 um particle size) column and isocratic mobile phase composed of 0.1% triethylamine pH 2.5 (adjusted with phosphoric acid) and acetonitrile mixture in a ratio of (70:30; v/v). UV detection at 270 nm was employed for monitoring nitroxinil degradation behavior over a linearity range of 1–75 µg/mL. Plackett–Burman experimental design was adopted for robustness testing of the developed chromatographic method. LC-mass identification of nitroxinil’s hydrolytic and oxidative degradations was attempted, and the suggested mechanism was deduced. The proposed method was successfully applied in determination of the drug in raw material and pharmaceutical dosage form.