A new method based on high performance liquid chromatography-electrospray ionization time of flight-mass spectrometry (HPLC-ESI-TOF/MS) was developed for the rapid identification of active compounds in Styela clava and the development of its specific chromatograms. Samples were extracted by ultrasonic-assisted extraction, and the extraction conditions were optimized. The developed HPLC-ESI-TOF/MS method was used to identify the components in Styela clava extract, and a specific chromatogram based on HPLC analysis was established. Ten compounds in Styela clava extract have been primary identified by HPLC-ESI-TOF/MS on-line detection combined with literature review. The result of similarity evaluation for specific chromatograms indicated that the quality of different Styela clava samples was not entirely consistent. This method has the advantages of simple operation, rapid measurement and it is a powerful tool for identification of active components in Styela clava and its quality control.