Abstract: The blood-brain barrier (BBB) is a critical biological structure which maintains the bathing microenvironment of brain but prevents the therapeutic drugs for central nervous system (CNS) work at the same time. Traditional Chinese medicine (TCM) shows advantages of fewer adverse reactions, mild and long-lasting effects in treating CNS diseases, and screening brain-penetrable constituents from TCM is a promising way for neuroprotective drug discovery. The purpose of the research is to study the material basis on the protective effect of Punica Granatum L. on CNS by combining sequential metabolism with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Many in situ models have been developed and widely used to study drug intestinal absorption, such as in situ closed-loop method, intestinal single-pass perfusion, intestinal recirculating perfusion, and intestinal perfusion with venous sampling (IPVS) method. In this study, the plasma samples of intestinal wall metabolism and intestinal flora metabolism were collected by intestinal perfusion and in situ closed-loop method, respectively, and the protoplasmic components and metabolites in the plasma sample were identified by UPLC-MS/MS after the extract of Punica granatum L. was prepared. Then, components in plasma were identified after the rats of comprehensive metabolism group were given the extract by intragastric administration. Finally, the components into the brain were found in the cerebrospinal fluid. A total of 38 chemical components were detected in the extract, including tannins, flavonoids, phenolic acids, and isocoumarins. The situations for intestinal wall metabolism and intestinal flora metabolism groups were similar, as the same 30 protoplasmic components and 12 metabolites were found. For the comprehensive metabolism group, 7 protoplasmic components and 18 metabolites were detected. 2 protoplasmic components and 4 metabolites were found in cerebrospinal fluid samples, including citric acid, ellagic acid, and two methyl ellagic acid isomers, dimethyl ellagic acid, and dimethyl ellagic acid glucuronide. The components into the brain of Punica Granatum L. and its metabolites were identified using multicomponent sequential metabolism, which provided evidence to clarify the protective effect of Punica Granatum L. on central nervous system. This study provides evidence that the intestinal perfusion and in situ closed-loop method can help filter out part of the unabsorbed components and reduce the workload of comprehensive metabolic component identification, and it is also a supplement to the missing components in the plasma of gavage group. In a word, compared with detecting the components in cerebrospinal fluid directly, the addition of multicomponent sequential metabolism can improve identification speed and accuracy greatly.