In this paper, a facile extraction strategy is reported for the analysis of isopentenyl pyrophosphate, a key isoprenoid, based on magnetic core-shell microspheres with Ti4+ ion exterior walls coupled with liquid chromatography and tandem mass spectrometry. Because of their excellent hydrophilicity and biological compatibility, the polydopamine@Fe3O4-Ti4+ microspheres display ideal isopentenyl pyrophosphate extraction efficiency. The technique includes three steps: sample loading, nonphosphate washing, and phosphate elution. Moreover, the microspheres can be regenerated by thorough washing with a specific solvent and can be reused multiple times. The liquid chromatography with tandem mass spectrometry separation was performed on aWelch Ultimate r XB-C18 column with a total chromatographic analysis time of 5 min; the analytical recovery was 98.52%. The proposed method was used to determine the isopentenyl pyrophosphate concentration in rat plasma samples collected from the Shanghai Chest Hospital. The results indicate the prospective value of the as-made microspheres for the sensitive and selective enrichment of phosphate compounds in complicated matrices.