Separation of equally sized particles distinguished solely by interfacial properties remains a highly challenging task. Herein, a particle fractioning method is proposed, which is suitable to differentiate between polymer‐grafted microparticles that are equal in size. The separation relies on the combination of a pressure driven microfluidic flow, together with simultaneous light illumination and temperature control. Heating the solution forces thermo‐responsive surface grafts to undergo a volume phase transition and therefore locally changing the interfacial properties of the microparticles. Light illumination induces the phoretic/osmotic activity of the microparticles and lifts them into a higher plane, where hovering particles experience a different shear stress proportional to the height. The light‐induced hovering height depends on the interfacial properties, and this complex interaction leads to different movements of the microparticles as a function of their surface grafting. The concepts are visualized in experimental studies, where the complex physical principle provides a simple method for fractioning a binary mixture with at least one thermo‐responsive polymer graft.
