Abstract: A rapid analysis methodology for ten neurotransmitters in serum was developed using ultra performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). A typical protein precipitation (PPT) method was employed for removing protein macromolecular compounds and efficient extraction of the studied compounds. A home-made accurate mass database (mass-to-charge (m/z), retention time, isotopic distribution and characteristic fragment ions) and MS/MS spectra library were used for accurate identification. Quantitative analysis was performed using the extracted m/z of full-scan MS and external standard method. Matrix effect was 62.4%-112.2% and the relative standard deviations (RSDs) were 1.9%-10.1%. The extracted recovery and RSDs were 73.6%-104.2% and 2.6%-10.6%, respectively. Experimental results indicated that good linearities (R²＞0.99) were observed in the respective concentration ranges. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.05-11.63 nmol/L and 0.09-46.50 nmol/L, respectively. Stability of standard solutions, quality control samples and serum samples can meet the requirements of biological samples analysis. Accuracy of the dilution effect (DE) was 5.1%-10.6% and the precision was 1.1%-5.3%, all within the range of ±15%. Carry-over effect (COE) was 0.4%-4.6% through the analysis of initial mobile phase after three consecutive injections of samples of high concentration. Moreover, this method was applied to the research of neurotransmitters metabolism in the serum of rats with ionizing radiation-induced brain injury. Results demonstrated that ionizing radiation causes neurotransmitter metabolism in serum, and can provide important references for the screening of new biomarkers in serum.