The accurate determination of adsorption isotherms is of key importance in the design and optimization of preparative chromatographic bulk separations and purifications. Issues to be considered involve other things: limited amount of test material, particularly in the case of the development of drugs; multicomponent mixture with widely different concentration levels of the main product and of the impurities. In this work, a rational procedure for the characterization of adsorption equilibria in reversed phase chromatography is proposed. This is applied to two different model separations, both involving an ascomycin derivative of industrial interest. The procedure is based on the peak fitting method and the use of a lumped pore diffusion model to simulate column dynamics. The accuracy of the obtained results is assessed thoroughly.