Abstract: To study the substances in fudosteine, one synthetic by-product and five forced degradation products were detected by hydrophilic interaction chromatography (HILIC). Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) was used for accurate mass determination and product ion scanning. Five related substances were identified in the products of mass spectra fragmentations elucidation, and verified further according to synthetic process and stress testing results. The results obtained are valuable for fudosteine manufacturing process control and quality assurance.