Following the underlying principles of quality by design mentioned in the ICH Q8 guidance, systematic approaches for the control of process-related impurities have been taken in the manufacturing process of fasudil hydrochloride, a potent Rho-kinase inhibitor and vasodilator. Three related impurities were found in fasudil hydrochloride lab samples by a newly developed RP-HPLC with volatile mobile phase gradient elution and UV detection method. The elemental compositions of the impurities were determined by positive ESI high-resolution TOF-MS analysis of their [M + H](+) ions and their structures were identified through the elucidation of the product mass spectra obtained by a triple quadrupole mass spectrometer. The key impurity was further verified through synthesis and organic spectroscopy including NMR and IR spectroscopy. The origins of these impurities were located and the effective approaches to eliminate them were proposed based on the redesign of the synthetic conditions. The results obtained are important for quality control in the manufacture of fasudil hydrochloride bulk drug substance and injection.