Objective?To establish a electrospray ionization-tandem mass spectrometry（ESI-MS／MS）method for the determination of glutamine（Gln）and glutamate（Glu）in dry blood spots，and provide its feasibility for screening of ornithine transcarbamylase deficiency and carbamoyl phosphate synthetase deficiency in neonatals.Methods?The dry blood spots samples were collected from newborn during the period from June to September，2020.The dry blood spots samples were processed with Non-derivatized MS／MS kit.Following the routine MS／MS detection，the residual sample was used for the detection of Gln and Glu.The residual samples were dissolved in extraction solution，then were detected by ESI-MS／MS with positive ion scanning mode.The accuracy，precision，stability of the method were verified and clinical application and evaluated.Results?The recovery and accuracy of Gln were good in the range from 125 to 1 250 μmol／L（recovery：98.01% to 107.24%，bias：0.56% to 4.8%）.The recovery of Glu was good in the range from 250 to 1 250 μmol／L（recovery：102.06% to 113.65%）.The accuracy of Gln was good in the rarge from 250 to 1 250 μmol／L（bias：0.09%～7.67%）.The stability test confirmed that no statistically significant difference of Gln levels before and after reconstitution was found，but the difference of Glu levels was statistically significant before and after reconstitution.Combined with the results of Gln and Glu in 6 ornithine transcarbamylase deficiency positive dry blood spots samples，the diagnostic accuracy was 100%.The reference intervals of Gln and Glu were set as＜106.63 μmol／L and＜188.24 μmol／L respectively.Conclusion?The research successfully established a method for detecting Gln and Glu by ESI-MS／MS in DBS，which is simple，low-cost and without interference in routine clinical detection.