Analytical separation parameters of a side-capped octaphenyl double-decker shaped silsesquioxanes (DDSQ) mixture with zero, one, and two hydroxyl groups were obtained with HPLC. These parameters were extrapolated for a large scale preparative separation. The frontal analysis was experimentally performed for each component in the mixture to obtain linear adsorption isotherm parameters. The slopes of the linear isotherms were related with the experimental HPLC retention times in a linear function to predict separation for other mixtures with similar characteristics. The HPLC chromatograms were simulated in ASPEN chromatography with acceptable accuracy. Parameters of the scale-up separation were determined and prediction of collection times for each compound in three different mixtures was evaluated by separations using 5 g scale. Most importantly, scale-up isolation of chemically asymmetric DDSQ structure was demonstrated.