The plasma membrane and the cell cortex are essential parts of the eukaryotic cell. The plasma membrane delimitates the cell and mediates communication with the outside. The cell cortex is the submembrane cytoskeleton shaping the cell and is able to reorganize for the passage of material. To study events at and near the plasma membrane, cryoelectron microscopy (cryo-EM) may be used. Most intact cells are too thick for direct cryo-EM imaging. Generating cell-free membrane patches could be a means to study features at the plasma membrane. Here we present an unroofing method, termed iMEM (isolation of membrane patches for cryo-EM) where the plasma membrane is isolated directly on an EM grid. The in situ isolation of membrane patches has several advantages: it is a one-step procedure providing a higher throughput than focused-ion beam cryomilling. It enables the time-precise control over biochemical events before cryofixation.