Abstract: Radix Ophiopogon, a kind of traditional Chinese medicine, is the root of Liliaceae Ophiopogon japonicas (Thunb.) Ker Gaw l. Modern research demonstrates that Ophiopogon japonicas can be used as a folk food medicine in free radical scavenging, reducing blood sugar, sedative hypnotic and anti-myocardial ischemia and arrhythmia, and so on. The major chemical compounds of this plant are mainly homoisoflavonoids, steroidal saponins and polysaccharides. According to the relevant reports, the homoisoflavonoids is a kind of specific compounds in Ophiopogon japonicas. However, due to the large number of different types of homoisoflavonoids in the herbal medicine, the structures are too similar to be distinguished from each other. Meanwhile, the differences of their relative contents in Ophiopogon japonicas are great. In this paper, the rapid separation ability of UPLC-LTQ-Orbitrap and the higher energy collision induced dissociation mode (HCD mode) were used to acquire high resolution fragment ion information and studied on the fragmentation patterns of homoisoflavonoids. The chromatographic separation of Ophiopogon japonicas extract was used Waters Acuqity UPLC^TM BEH C18 column, mobile phase was 0.05% formic acid in water and acetonitrile system, and the chromatographic conditions for the rapid separation were optimized. To avoid interference with other components and obtain the tandem mass spectrometry high content and low content data, a method based on parent ions detection list in the negative ion mode detection was established. Furthermore, the established method was helpful in investigating the dissociation pathways of homoisoflavonoids in different HCD cleavage energy and confirming the characteristic fragment ions for the identification. Meanwhile, 68 constituents attributed to homoisoflavonoids in Ophiopogon japonicas are finally identified, including 26 homoisoflavonoids, 31 homoisoflavanones, and 11 homoisoflavonoids glycosides. The results indicate that the HCD model can provide abundant and stable information of fragment ions, which can be adopted to quickly and accurately clarify the homoisoflavonoids in complex system. This study can provide an effective analytical method to explore the material basis of sequential constituents in traditional Chinese medicines.