The present study aims to compare the phenolic profiles of organic and conventional tomatoes bought in the market. For the quantification and identification of individual polyphenols, liquid chromatography coupled to mass spectrometry in tandem mode (LC-MS/MS) was carried out. Confirmation of the compounds previously identified on the triple-quadrupole was accomplished by injection in the high-resolution system (QToF-MS). In this way, 34 compounds were identified in tomato fruits. Recoveries of targeted polyphenols exceed 78% for conventional and organic tomatoes, respectively. The method intraday precision ranged between 3 and 5%, whereas the interday one was below 12%. Comparing the analyses of tomatoes from conventional and organic production systems demonstrated statistically higher levels (P < 0.05) of phenolic compounds in organic tomatoes. This methodology allowed finding differences in the bioactive components of organic and conventional tomatoes not previously reported.