Technical Abstract: A study of the mass spectroscopic behaviors of anthocyanins in the negative ionization mode was reported and it can be used for differentiation anthocyanins from other non-anthocyanin polyphenols. For the study, an ultra-high performance liquid chromatography with high resolution mass spectrometry (U-HPLC-HRMS) method was developed. Traditionally, anthocyanin analysis in mass spectrometry is carried out in the positive ionization mode only. The new strategy found , in the negative ionization mode, a series of characteristic ions, e.g. [M-2H]-, [M-2H+H2O]- , formic acid adducts, and double charged ions were observed for the MS analysis of anthocyanins. The characteristic ions can be used for identification and differentiation of anthocyanins and non-anthocyanin phenolic compounds. Comprehensive studies were performed on the differentiation of anthocyanins and non-anthocyanin polyphenols in blue berry, Brassica compestris L. var. purpurea Bailey, and red radish. Tentative identification of these phenolic compounds was made by accurate mass measurement with <3 ppm accurarcy and multiple-stage mass fragmentation data in both the negative and positive ion in one single LC run. The whole cycle time of the new method is 1.8 sec for 2 full scans and 6 data-dependent scans. The data generated from a single LC run enables rapid and reliable differentiation and identification of anthocyanins and non-anthocyanins in botanicals and foods.