Abstract: The study aimed to establish a HPLC method and a two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS) method for the determination of the related substances degradated by tryptophan in pediatric compound amino acid injection and the exploration of the degradation pathway of tryptophan. The HPLC method was carried out on a Waters Atlantis T3 C18 (250 mm × 4.6 mm, 5 μm) column by gradient elution using phosphate buffer solution-acetonitrile (990∶12) and phosphate buffer solution-acetonitrile (65∶35) as the mobile phases to separate tryptophan and the related substances degradated by tryptophan. The selected components were then trapped in switch valve tube lines respectively and delivered to the second-dimensional desalting and deacidification gradient elution which was performed with an Agilent ZORBAX Eclipse Plus C18 (50 mm × 3.0 mm, 1.8 μm) column using acetonitrile-water (95∶5) as the mobile phases. The positive ion mode of the electrospray ionization source was adopted, the parent ions were detected using MS scan mode and the fragment ions were detected using targeted MS/MS mode. The structure of the unknown impurities was figured out by mass spectrometry elucidation. The results showed that tryptophan and its related substances in pediatric compound amino acid injection can be adequately separated under the established analytical conditions. A new impurity produced by the interaction between tryptophan and antioxidant excipients such as sodium bisulfite or sodium metabisulfite was discovered for the first time. The method can provide reference for the quality control of this injection.