Rationale A series of photodegradation impurities and a series of degradation impurities produced in autoclaving in xinfujunsu injection were discovered, and these unknown impurities were separated and characterized thoroughly using liquid chromatography tandem quadrupole time‐of‐flight mass spectrometry. Methods The column was a Platisil 5 μm ODS (4.6 × 250 mm, 5 μm). For the analysis of degradation impurities caused by light irradiation and autoclaving, the mobile phase was composed of 0.01 M ammonium formate aqueous solution and acetonitrile/isopropanol (90:10, V/V). Full scan LC–MS and LC–MS2 was carried out to obtain as much structural information as possible. The fragmentation behavior of actinomycin D, actinomycin S3, and its impurities was studied and used to obtain information about the structures of these impurities. Results Based on MS2 spectral data and exact mass measurements, the chemical structures of two series of degradation impurities were characterized, among which five unknown impurities were photodegradation impurities and seven unknown impurities were degradation impurities produced in autoclaving of xinfujunsu injection. Conclusions Based on characterization of impurities, this study also revealed the cause of impurity production and provided guidance for enterprises to improve the process and drug packaging material to reduce impurity content. Furthermore, this study also provided scientific basis for further improvement of official monographs in pharmacopoeias.