Poly-/Oligo(phenylene ethynylene)s (PPE/OPEs) possess conjugated, rigid backbones and are of considerable interest due to their unique photophysical and electronic properties. In this study, we introduce a photoswitchable azobenzene moiety into sequence-defined OPE backbones to investigate light-induced conformational changes in well-defined molecular scaffolds. A series of three azobenzene-functionalized oligomers was synthesized and characterized using a combination of established analytical techniques and SEC. Owing to the geometric constraints and rigidity of the OPE framework, the impact of E/Z photoisomerization on the overall molecular size is significantly amplified. This enabled, for the first time, a quantitative analysis of the photoresponse via SEC, which revealed distinct changes in the hydrodynamic volume of the oligomers upon irradiation. These findings offer deeper insights into the design and behavior of stimuli-responsive macromolecular materials and demonstrate the utility of SEC as a complementary analytical tool. This approach also helps to overcome certain limitations of conventional techniques such as 1H NMR spectroscopy and HPLC in monitoring dynamic structural transformations of macromolecules.