Miniaturization of analytical instruments has attracted a wide interest in analytical chemistry over the past decade because of the advantages of reduced reagent consumption, better analytical performance, and shorter analysis time. The widespread interest in this field has resulted in efforts to develop chips. For chips involving separation, injection is a key step to achieve efficient and sensitive analysis. This work presents a comparative study of two electrokinetic injection modes in chips: the floating, which has been mainly used up to now, and the dynamic. This study was done with a crossjunction, either with numerical simulations or with experiments. Experiments were carried out with homemade PDMS-glass microsystems involving zonal electrophoresis analysis of five derivatized amino acids. Injected amount, reproducibility, separation efficiency, and analyte discrimination were evaluated and discussed. The experimental results were successfully correlated with numerical simulations. It appeared that the dynamic injection mode is much more appropriate than the floating mode as it is faster (reduction by a factor 2 of the total analysis time here), more reproducible (RSD of peak areas equal to 1.3% (n = 4) instead of 10% (n = 4)), and leads to more efficient separation (about 20% with 3 cm separation channel length) for the same injected amount, whatever the amount, because the sample plug is less dispersed.