An optimized method was developed for simultaneous determination of melamine (MEL) and cyanuric Acid (CYA) in meat, liver and kidney tissues of five kinds of animals (cattle, goat, pig, chicken and fish). MEL and CYA were extracted with acetonitrile/water (70/30) by shaking. After being simply defatted with n-hexane, both analytes were detected within 12 min using liquid chromatography-triple quadrupole mass spectrometry (LC/MS/MS) on Waters Atlantis HILIC columns. The optimized method using isotope-labeled analytes as internal standard was linear over a range of concentrations with 2-500 ng/mL for MEL and 4-500 ng/mL for CYA. The mean recoveries of MEL and CYA in tissues respectively ranged from 83-108% and 75-102% with RSD < 11%. The limits of detection (LOD) for MEL and CYA respectively were 0.01 and 0.02 mg/g of tissues. The results showed that the optimized method have a simple procedure of sample preparation, a wide linear range and higher sensitivity, and therefore suit for determination of MEL and CYA in animal tissues especially when analyzing big batch of samples from pharmacokinetic studies of MEL and CYA in animal.