Polymer membranes provide a highly promising platform for the development of an efficient and sustainable technique for separation. Ideally such membranes combine a high flux with a high selectivity requiring thin defect-free membranes. The layer by layer (LBL) assembly technique has proven to be a versatile and simple method for the fabrication of very thin polyelectrolyte multilayers making it highly suitable for the preparation of separation membranes. Recent developments in this field related to membrane preparation and their applications in separation processes are presented and discussed in this review. An overview of the different fabrication techniques of such membranes will be first provided. In addition, the formation mechanism and the parameters that can be varied to tune the properties of the membranes will be discussed. Finally, the potential applications of these membranes in different separation areas such as pervaporation, nanofiltration, solvent resistant nanofiltration, reverse osmosis, gas separation and forward osmosis will be addressed