Amino metabolites are important compounds that play a key role in plant growth and development. A metabolic profiling analysis method of amino metabolites in plant extract was developed based on pre-column derivatization-ultra high performance liquid chromatography- mass spectrometry. Using the tobacco leaf as an example, a total of 87 amino metabolites, including amino acids, amines, peptides, alkaloids etc. were detected. The repeatability of the method was good with RSDs of 85 amino metabolites between 1. 5% and 18. 8%. Forty-three amino metabolites validated by standard samples showed good linearity with the correlation coefficients of 0.993-0.999, covered linear range of four orders of magnitude. The limits of detection were 0.03-6.58 ng/mL. The intra-day and inter-day precisions were 0.7%-15.6% and 0.8%-22.9%, respectively. The recoveries were 74.4%-122.7%. The influence of topping on metabolic profiling of amino metabolites in fresh tobacco was investigated using the developed method. The results showed that the amino metabolites in the upper tobacco leaves were most affected than those in the middle and lower leaves. Metabolism of amino metabolites in the upper leaves after topping was mainly towards the alkaloid synthesis. The method integrated the advantages of triple quadrupole mass spectrometry and high resolution quadrupole-time of flight mass spectrometry. It can be used for metabolic profiling analysis of amino metabolites in plant extract with high sensitivity and selectivity.