Abstract High-performance liquid chromatographic (HPLC) was developed for fingerprint analysis of Pseudostellaria heterophylla (Miq.) Pax. Liquid chromatography–electrospray ionization-time-of-flight mass spectrometry (LC–TOF-MS) technique was first employed to identify the components of the fingerprint. Twelve major peaks in chromatographic fingerprint were analyzed by on-line LC–TOF-MS analysis; one cyclic peptide was unequivocally identified and five cyclic peptides were tentatively assigned based on their MS data. These cyclic peptides served as the marker peaks in the HPLC fingerprints. The chromatographic fingerprints have been analyzed by similarity index calculations and hierarchical clustering analysis (HCA). The result showed that the HPLC fingerprints could be used to determine the optimal harvest time for P. heterophylla (Miq.) Pax and to authenticate the species of the herb.