A new chromatographic response function (CRF(2D)) is proposed and tested for the estimation of the quality index of separation in comprehensive two-dimensional liquid chromatography (2D-LC) of complex organic mixtures. This objective function is based on the concept of peak purity for one-dimensional liquid chromatography, which has been redefined for 2D-LC. The new CRF(2D) also includes other separation quality criteria, namely the number of 2D peaks appearing in the chromatogram and the analysis time. To compute the peak purity for a given 2D peak, three important steps have been tackled in this study: (a) the development of an alternative algorithm for detecting 2D peaks automatically from real experimental 2D-LC data; (b) the application of a mathematical model to fit the obtained chromatographic data; and (c) the estimation of the volume of the overlapping region between two or more 2D peaks. The performance of the developed CRF(2D) was compared to that of an existing resolution measure, using simulated chromatograms. The capability of the new function to qualify the overall separation degree that it is attained under different chromatographic conditions was further assessed through a 2D-LC study of a mixture of four aromatic compounds.