Triptolide (TP) is one of the most important biologically active components of the Chinese herb Tripterygium wilfordii Hook f (TWHf). A novel high-performance liquid chromatography/mass spectrometry (LC/MS) method was developed to study the kinetic release of TP after intravenous injection of the newly synthesized 14-succinyl triptolide-lysozyme (TPS-LZM). The method was validated in terms of selectivity, linearity, precision, accuracy, stability, limit of detection (LOD) and limit of quantification (LOQ). The calibration curve was linear over the concentration range 0.5-400.0 ng/g. The mean recovery of triptolide from spiked samples, in a concentration range of 0.5-400.0 ng/g, was 91.84% (RSD = 3.69%, n = 3). The intra- and inter-assay coefficients of variation were less than 6.38%. The method with simple sample pretreatment and being highly specific and precise, can be used for analysis of triptolide release in rat kidney after intravenous injection of renal-targeting TPS-LZM conjugate. The results showed that, as compared with free TP, TPS-LZM could significantly increase the concentration and prolong the action time of TP in the kidney.