ObjectiveTo rapidly separate and identify the chemical components in traditional Chinese herbal medicine of Oldenlandia diffusa and its injection preparations by high performance liquid chromatography-time of flight mass spectrometry (HPLC-TOFMS). MethodsAn Agilent Zorbax XDB-Cl8 column (250 mm×4.6 mm, 5 μm) was used for separation and identification of chemical components in Oldenlandia diffusa, with a mobile phase of 0.3% acetic acid (A) and methanol (B) in gradient elution, 0-30 min, 30%-90%B. The flow rate was set at 1.0 ml/min and the injection volume was 10 μl. The time-of-flight mass spectrometer was equipped with an EIS ion source. The scanning mass range was between m/z 100-1 000. ResultsThe traditional Chinese medicine of Oldenlandia diffusa and its injection preparation were on-line separated and characterized by HPLC-TOFMS, and 11 chemical compounds were identified in Oldenlandia diffusa, 6 compounds in market injection preparation, and 2 compounds in the injection prepared by ourselves.ConclusionChromatographic demonstration of chemical compounds in Oldenlandia diffusa in one run provides a foundation for the further studying the metabolism and mechanism of Oldenlandia diffusa and its injection preparations.