Abstract: Internal extractive electrospray ionization mass spectrometry (iEESI-MS) was employed to directly analyse the choline, fructose and sucrose in four kinds of red peppers under positive ion detection mode in the mass range of m/z 50-1 000. Changes of the chemical constituents in red pepper during storage period were directly followed, and principal component analysis (PCA) was used to differentiate freshness of red pepper. The result indicates that the relative abundance ratios of choline to fructose and sucrose to fructose in red peppers are linearly decrease during the storage at 25 ℃ and humidity of 60%, and the corresponding decline curves which associate with the metabolism of the red pepper are plotted. Based on the ratio of choline/fructose or sucrose/fructose, the freshness and quality of the red pepper can be identified by iEESI-MS at the molecular level. In comparison with other methods for the analysis of fruits and vegetables, the method shows the merit of such as minimum sample pretreatment, simplicity and rapid analysis, which has promising applications in food safety and quality control analysis.