In this work, capillary isotachophoresis (ITP) was used to determine the effective charge of the first five generations of dendrigraft poly-L-lysines. This approach, which is based on the linear dependence of ITP zone length of the solute on its concentration and effective charge, offers a simple and straightforward method for effective charge determination. The cationic ITP system employed in this work yields good linearity, repeatability and sharp zones. The value of effective charge number per one lysine residue obtained for long linear poly-L-lysine is in a good agreement with the Manning theoretical value (0.5). Results obtained for dendrigraft poly-L-lysines show a dramatic decrease in the effective charge number per lysine residue with increasing generation number, from 0.84 for short oligolysines (generation 1) down to 0.08 for the fifth generation. This decrease in effective charge is due to the proximity of charged groups in the dendrigraft structure of higher generation number.