• Enantioseparation of metalaxyl and metalaxyl acid was realized in reverse phase conditions. • A LC-MS/MS for simultaneous determination of enantiomer residues of metalaxyl and metalaxyl acid. • Mixed anion exchange solid-phase extraction was used for sample preparation and clean-up. • The LOQs were 0.3–1.0 μg/kg in four animal muscle tissues. The toxicity of the two enantiomers of metalaxyl is significantly different and, for food safety concerns, stereoselective analysis of its residues is extremely important in animal muscle tissues. Herein, a simple, sensitive and reproducible method for the simultaneous stereoselective analysis of metalaxyl and its metabolite metalaxyl acid in animal-derived foods has been established based on mixed-mode anion exchange solid phase extraction and chiral liquid chromatography-tandem mass spectrometry. Under the optimized separation and sample pretreatment conditions, good linearity (r > 0.99) was obtained for four target analytes in four animal muscle tissues including bovine, chicken, swine and fish. Mean recoveries of all enantiomers in muscle tissue samples were 89.5–110.3 % with relative standard deviations below 12.4 %. The limits of quantification of the analytes were 0.3–1.0 µg/kg. The proposed method provides an effective means to monitor the enantiomer residues in real samples whether using highly active R -metalaxyl or racemic mixture drugs. [ABSTRACT FROM AUTHOR]