As a preceding study for investigating the influence of sound wave stimulus on Arabidopsis thaliana metabolomics, the polar secondary metabolomes of the plant were determined using high performance liquid chromatography coupled with tandem mass spectrometry. A total of 10 polar secondary metabolomes were characterized and quantified. Among them, 4 metabolomes, p-coumaroylagmatine isomer (7 and 8), p-coumaroylagmatine isomer (9 and 10) were identified in the plant for the first time. The validation was conducted in terms of linearity, recovery, precision, limit of detection (LOD) and limit of quantification (LOQ). The validated method was applied to the simultaneous quantification of the 10 polar secondary metabolomes.