Rapid extraction, discrimination and quantification of isomers, especially for thermally unstable isomers, in complex natural products by direct MS are of great challenge due to the time-consuming sample pretreatment procedures, thermal instability, and identical mass behaviors. This work developed an online extraction-quadrupole time-of-flight tandem MS (OLE-QTOF-MS/MS) system for quantification of isomeric phenylethanoid glycosides (acteoside and isoacteoside; acteoside isomerizes to isoacteoside when heated) in Clerodendrum bungei within 1.5 min. Solid C. bungei was packed into a guard column, which was then positioned on the manual injection valve for OLE-QTOF-MS/MS analysis. The relative intensity ratio of diagnostic ion pairs (I461/I161) fragmenting at the heterogeneous linkage site changed linearly with the relative content of acteoside from 0% to 100% (R2 = 0.9991), and then the content ratio of acteoside and isoacteoside in C. bungei was determined as 85.3 : 14.7 (m/m) with an RSD of 1.7%. Finally, the absolute quantification results (acteoside, 17.5 ± 0.8 mg g−1 and isoacteoside, 3.0 ± 0.8 mg g−1) were obtained through a standard addition method with RSD < 3.4% for intra- and inter-day variations. Results demonstrated that OLE-QTOF-MS/MS was a promising and practical method for simple, rapid and effective extraction and analysis of isomeric compounds in complex matrices.