Eukaryotic cells utilize sub-cellular compartmentalization to restrict reaction components within a defined localization to perform specified biological functions. One way to achieve this is via membrane enclosure; however, many compartments are not bounded with lipid membrane bilayers. In the past few years, it has been increasingly recognized that molecular components in non- or semi-membrane-bound compartments might form biological condensates autonomously (i.e., without requirement of energy input) once threshold concentrations are reached, via a physical chemistry process known as liquid–liquid phase separation. Molecular components within these compartments are stably maintained at high concentrations and separated from the surrounding diluted solution without the need for a physical barrier. Biochemical reconstitution using recombinantly purified proteins has served as an important tool for understanding organizational principles behind these biological condensates. Common techniques include turbidity measurement, fluorescence imaging of 3D droplets, and atomic force microscopy measurements of condensate droplets. Nevertheless, many molecular compartments are semi-membrane-bound with one side attached to the plasma membrane and the other side exposed to the cytoplasm and/or attached to the cytoskeleton; therefore, reconstitution in 3D solution cannot fully recapture their physiological configuration. Here, we utilize a postsynaptic density minimal system to demonstrate that biochemical reconstitution can be applied on supported lipid bilayer (SLB); we have also incorporated actin cytoskeleton into the reconstitution system to mimic the molecular organization in postsynaptic termini. The same system could be adapted to study other membrane-proximal, cytoskeleton-supported condensations.