To analyse and evaluate the uncertainty in the determination of neotame content in roasted seeds and nuts by UPLC-MS/MS. A mathematical model of uncertainty in the determination of neotame content in roasted seeds and nuts was established, various factors of uncertainty in the whole determination process were studied and evaluated, the components of uncertainty in the method were confirmed and composed. The results showed that the expanded uncertainty was 0.71 mg/kg(k=2) when the neotame content was 7.69 mg/kg, which was greater caused by preparation of standard stock solution and repeatability of determination.