Capillary isotachophoresis (ITP) in cationic regime of the separation with conductometric detection has been used for the separation and determination of basic amino acids (arginine, histidine, and lysine) in pharmaceutical preparations. Several electrolyte systems of different compositions and pH were examined. The optimized ITP electrolyte system consisted of 10 mmol/L potassium acetate adjusted to pH 4.0 with acetic acid as the leading electrolyte with the electroosmotic-flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC), and 10 mmol/L beta-alanine as the terminating electrolyte. The proposed electrophoretic method was successfully validated. It was convenient for the sensitive, simple, rapid and highly reproducible assay of these amino acids. Good quantitation was obtained in short analysis times (a single analysis took about 10 min). The minimal sample pretreatment and low running costs make the proposed ITP method a good alternative to commonly used analytical methods. The obtained results suggest that the proposed method is suitable for routine assay of basic amino acids in pharmaceuticals.