The mammalian plasma membrane is a fluid and highly functional semi-permeable barrier between the cell and its environment. It consists of proteins and lipids which are not homogeneously distributed, but show local and temporal heterogeneity. Different mechanisms are suspected to be responsible for membrane heterogeneity, such as lipid-lipid and lipid-protein interactions. In addition, the cortical actin cytoskeleton has been proposed to restrict the motion of proteins and even form diffusion barriers to lipids. Such corrals created by actin are thought to be very small – around 100 nm – making them hard to address experimentally. The aim of this work was to establish a method capable of testing the role of actin in the diffusion behavior of lipids and membrane proteins. For this, a protein micropatterning approach to reorganize the cortical actin skeleton of live cells was used, allowing the measurement of protein and lipid mobility as a function of the local actin density.