In the last decade the simulated moving bed (SMB) process was implemented successfully in the pharmaceutical industry to perform difficult separations of valued added products. Mainly liquid solvent mixtures with adjusted but constant composition were applied as the mobile phases (isocratic operation). Only recently the potential of modulating the solvent strength during the process was realised (gradient operation). The purpose of this paper is to present the results of studying a two-step gradient SMB process using a weaker solvent to introduce the feed and a stronger solvent for efficient adsorbent regeneration. A mathematical model for the simulation of this process was proposed and solved numerically. Systematic calculations were performed in order to identify suitable operating conditions as a function of averaged desorbent concentrations taking into account the locally changing nonlinear adsorption equilibria. Based on the predictions and on preliminary experimental investigations devoted to determine the required model parameters the separation of a mixture of two cycloketons using normal-phase silica was designed. A two-step gradient SMB process was applied using pure n-hexane as the weak solvent in the feed stream and pure ethyl acetate as the strong solvent in the desorbent stream. The achieved purities and concentrations in the raffinate and extract streams confirmed the potential of the gradient technique applied. In agreement with the theoretical results a significant improvement in the process performance compared to the common isocratic SMB separation was found.