The synthesis routes in the production of polysilsesquioxanes have largely relied upon in situ formations. This perspective often leads to polymers in which their basic structures including molecular weight and functionality are unknown [ Lichtenhan , J. D. ; et al. Silsesquioxane-siloxane copolymers from polyhedral silsesquioxanes Macromolecules , 1993 , 26 , 2141 - 2142 , http://dx.doi.org/10.1021/ma0060a053 ]. For a better understanding of the polysilsesquioxane properties and applications, there is a need to develop more techniques to enable their chemical characterization. An innovative method was developed to determine the molecular weight distribution (MWD) of an oligosilsesquioxane synthesized in-house from (3-aminopropyl)triethoxysilane. This method, which can be applied to other silsesquioxanes, siloxanes, and similar oligomers and polymers, involved separation using high performance liquid chromatography (HPLC) and detection using mass spectrometry (MS) with electrospray ionization (ESI). The novelty of the method lies on the unique determination of the absolute concentrations of the individual homologues present in the sample formulation. The use of absolute concentrations is necessary in estimating the MWD of the formulation when relative percentage, which is based solely on mass spectral ion intensities, becomes irrelevant due to the disproportionate response factors of the homologues. Determination of absolute concentration requires the use of single-homologue calibration standards. Because of commercial unavailability, these standards were prepared by efficient fractionation of the original formulation.