Analytical isotachophoresis has proved to be an excellent method for separation and quantitative measurement of ionic species in biologic fluids. Isotachophoretic separation of cations in presence of a complex-forming nonionic detergent (e.g. Triton X-100) in 98% methanol enables simultaneous quantitative determination of ammonium, sodium, potassium, magnesium, and protonated creatinine in urine samples. For calcium determination in urine, lead acetate was used to precipitate anions that form complexes with calcium. The mean deviation from 100% recovery of ammonium, sodium, potassium, magnesium, calcium and creatinine added to urine samples is 3.1%. The proposed method shows good agreement with chemical (routine) methods.