Abstract: An ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of ADMA, SDMA, NMMA, L-Arg and Cit in human plasma after administration of ilaprazole. The plasma samples were prepared using protein precipitation by acetonitrile after addition of deuterated internal standard. The analytical column was Waters Atlantic HILIC (2.1 mm×50 mm×3 μm), and the mobile phase was composed of acetonitrile (containing 0.5% acetic acid and 0.025% trifluoroacetic acid)-water (containing 0.5% acetic acid and 0.025% trifluoroacetic acid) (85∶15, V/V). The flow rate was 0.25 mL/min and the sample run time was 3.0 min. A tandem mass spectrometer coupled with positive electro-spray ionization (ESI) source was used for detection. The quantitative analysis was performed on selective ion chromatograms acquired by a multiple reaction monitoring (MRM) mode of following transitions: ADMA (m/z 203.2→46.0), SDMA (m/z 203.2→172.1), NMMA (m/z 189.0→70.0), Cit (m/z 176.0→70.0), L-Arg (m/z 175.1→60.071), and D₇-ADMA (m/z 210.0→77.1). The specificity, accuracy, linearity, intra-day and inter-day precision, recovery and matrix effect were investigated in this study according to the guidance issued by the Food and Drug Administration (FDA) of USA. The method was validated over the concentration range of 0.1-5 mmol/L for ADMA, SDMA and NMMA, 10-250 mmol/L for L-Arg and Cit, respectively. Inter-day and intra-day precision were less than 15% and accuracy was within 85%-115%. The linear regression (weighed by 1/x²) was applied to establish the relationship between plasma concentration and peak area ratio of each analyte (minus that of the blank) to its IS. Topical equations of ADMA, SDMA, NMMA, L-Arg and Cit were as follows: y=0.385 1x+0.207 7 (r=0.996 7), y=0.418 0x+0.200 7 (r=0.998 3), y=0.366 2x+0.048 31(r=0.998 5), y=0.011 53x+1.204 9 (r=0.998 0), and y=0.043 21x+8.315 4 (r=0.994 1), respectively. The extraction recoveries of ADMA, SDMA, NMMA, Cit and L-Arg were (92.5±8.6)%, (88.5±6.5)%, (50.6±3.1)%, (27.0±0.3)% and (33.9%±2.1)%, respectively. The matrix effects about ADMA, SDMA, NMMA, Cit and L-Arg were (90.5±6.3)%, (83.0±3.5)%, (50.6±2.5)%, (41.0±0.1)%, and (32.9±0.2)%, respectively. Therefore, this method is rapid, simple and sensitive, and which can provide a highly efficient detection means to the future clinical disease diagnosis.