Objective To establish a method for simultaneous determination of melamine and cyanuric acid in rat by HPLC-MS/MS.Methods Using isotope-labelled melamine and cyanuric acid as internal standard,rat plasma and tissue samples were pretreated with dichloromethane-acetonitrile,and separated by chromatographic column switching technique.Detection was carried out by HPLC coupled with electrospray ionization(ESI) working on segment ESI-and ESI+ switching mode.Results The calibration curve of melamine and cyanuric acid were linear(r≤0.998) in the range of 0.3-75 μg/ml.The average recovery rates of melamine and cyanuric acid(added with 0.9,5.0 and 50 μg/ml) in rat plasma were 97.4%-99.4% and 96.8%-98.9%,respectively;while the relative standard deviations(RSD) of the method were 2.43%-6.35%(inter-day),1.70%-4.12%(intra-day),0.30%-9.17%(inter-day) and 0.75%-3.39%(intra-day),respectively.The average recovery rates of rat liver tissue(added with 0.9,5.0 and 50 μg/ml) were 90.8%-104.6% and 91.7%-106.0%,respectively;while the relative standard deviations(RSD) of the method were 3.70%-5.14%(inter-day),2.84%-4.50%(intra-day),1.31%-2.21%(inter-day) and 1.12%-3.36%(intra-day),respectively.Conclusion The method was simple in sample pretreatment,less matrix interference,efficient,precise,accurate and specific.It was ideal to study the mechanism of metabolic behavior and toxicology of melamine and cyanuric acid in animals.