Abstract Determination of optimal composition of a mixed mobile phase used in separation of different substances by adsorption TLC methods is performed usually by the experimental trial and error method. In this paper we have presented and discussed equations permitting to determine the relationships between RM values of the substances and the composition of binary and ternary mobile phases on the basis of measured chromatographic and adsorption parameters and experimentally determined partition coefficients of the substance between the mobile phase components. A good agreement between theoretical and experimental data has been shown. The results of these investigations allow for more precise prediction of the mobile phase optimal composition for the mixture separation.