This work aims at evaluating the effect of successive grinding and sieving processes on the polyphenol content of plants. Powders of particle size ranging from 20 to 500 μm and over were produced from aerial parts of Hypericum perforatum and Achillea millefolium. The evaluation of total phenolic content and antioxidant activity, as well as the identification and quantification of some bioactive compounds by LC-ESI/MS were performed. The highest antioxidant activity was obtained for the 100–180 μm fraction: IC50 of 0.43 and 0.51 mg/mL for H. perforatum and A. millefolium, respectively. LC-ESI/MS analyses evidenced that two intermediate granulometric classes, 100–180 μm and 180–315 μm, allowed achieving the highest polyphenol content. These results show that fine grinding and sieving lead to a differential distribution of bioactive compounds according to particle size.