Gut microbiota-host co-metabolites play an essential role in maintaining homeostasis, and their concentration changes are closely related to a variety of diseases. Developing a targeted metabolomics analytical platform for these co-metabolites will help to elucidate the relationship between intestinal flora and host. Here we present a simple and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of nine gut microbiota-host co-metabolites in rat serum, urine and feces. The compounds were separated on a reversed-phase C18 column using gradient elution with a solvent system consisting of methanol and water (containing 0.05% formic acid) and a 7-min run time. All of the calibration curves exhibited good linear relationships (R2 ≥ 0.9984, Percent Residual Accuracy ≥93.27%). The intra- and interday precision, expressed as relative standard deviation (RSD), was ≤ 14.84%. The accuracy was within 100 ± 13.16% for all analytes. The recovery of the nine compounds in biological samples was ≥ 85.80% with an appropriate RSD (≤12.04%). The validated method was successfully applied to monitor the global changes of these metabolites in obesity. Taken together, these results demonstrate that the method can simultaneously determine the nine co-metabolites in multiple biological matrices and is an essential part of the targeted metabolomics analytical platform, which may become an approach to evaluate the occurrence, development and therapeutic effects of metabolic diseases. Copyright © 2019 Elsevier B.V. All rights reserved.