Rationale Hirsutinolide-type sesquiterpene lactones (SLs) are natural biologically active compounds mainly found in the genus Vernonia. Very few studies have been published about the fragmentation mechanisms of SLs generally and none about hirsutinolides, although they have drawn attention through their biological and taxonomical interest. This work aims to propose a mass spectrometry fragmentation pattern for hirsutinolides in order to detect and to identify them in a botanical extract. Methods The fragmentation pathways of six pure hirsutinolides isolated from Pseudelephantopus spiralis were established by positive ion electrospray high-resolution linear ion trap Orbitrap tandem mass spectrometry (ESI(+)-HRMSn). A resolutive, hyphenated ultra-high-performance liquid chromatography (UHPLC) coupled to diode array detection (DAD) and ESI(+)-HRMSn method was then implemented to separate and analyze them. The ionization behaviour and diagnostic product ions were investigated by both methods. The UHPLC/DAD-ESI-HRMSn method was applied for the dereplication of a plant extract. Results For the six standard compounds, the main fragmentation pattern consists first in the loss of the side chain in the C-8 position followed by the loss of the substituent in the C-13 position. UHPLC/HRMS analyses of hirsutinolides mainly produced sodiated molecules or [M+H-H2O]+ ions. The high-abundance product ions at m/z 299 and 259 were established to be the characteristic diagnostic ions of the hirsutinolide core. The analysis of a P. spiralis extract further led to the identification of two putative hirsutinolides. Conclusions The UHPLC/DAD-HRMSn method combining characteristic fragmentation patterns and the profiles of the product ions generated in the MS and MS/MS spectra is an effective technique for characterizing hirsutinolide-type SLs.