A simple, sensitive and reliable method for the determination of valnemulin (VLM) in swine muscle, kidney and liver by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The analyte was extracted using the mixture of acetonitrile and 0.01mol·L-1 hydrochloric acid. The crude extracts were subjected to liquid-liquid partition with w-hexane followed by solid phase extraction using a mixed-mode strong cation-exchange column. The chromatographic separation was performed using a gradient on a Waters Symmetry Shield RP C18 column with a mobile phase of acetonitrile-water containing 0.1％ formic acid, and the analyte was detected by multiple reaction monitoring (MRM) using positive eletrospray ionization (ESI). The method has been validated at the limit of quantification (LOQ), at half of the MRL and at the MRL. The limit of detection (LOD) and LOQ of swine tissues were 1 ng·g-1 and 5 ng·g-1 respectively. The mean recoveries from fortified samples ranged from 84.2％ to 92.3％, with coefficients of variation ranging between 7.5％ and 15％.