A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in-capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC-MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC-MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC-MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.