A stepwise elution technique is developed to estimate the linear solvent-modulated isotherm parameters of taxanes from dilute plant tissue culture broth. The technique is proven to be robust under various experimental conditions because mass transfer effects are decoupled from isotherm effects on elution bands. Simple algebraic equations are derived to relate isotherm parameters to the retention times at different solvent compositions in stepwise elution under volume overload conditions. A minimum loading volume, which is a function of mass transfer parameters, column geometries, and adsorption affinities, is estimated from moment analysis to ensure that band spreading due to mass transfer does not affect the accuracy of this method. The isotherms of a low-cost solute, phenylalanine, under different ethanol concentrations are measured by a standard multiple frontal chromatography method. The results serve as benchmarks for the parameters that are estimated over the same ethanol concentration range using the stepwise elution technique. The parameters determined from these two independent methods are in close agreement. For paclitaxel and cephalomannine, the retention times and peak shapes from plant tissue culture broth experiments are well predicted from rate model simulations using the parameters estimated from single-component elution data.