Real-time membrane integrity monitoring is critically needed in order to ensure that reverse osmosis (RO) treatment and desalination processes provide a reliable barrier against the passage of pathogens for potable reuse applications. A pulsed marker membrane integrity monitoring (PM-MIM) method was developed for real-time assessment of RO membrane integrity. The sensitivity of the PM-MIM method was evaluated, using a fluorescent molecular marker, in a plate-and-frame RO (PERO) membrane system with intact and compromised membranes (induced mechanically and via chemical oxidation). Analysis of the marker permeate concentration time profiles, in response to a controlled marker feed pulse, allowed unambiguous detection of marker enhanced passage, which was indicative of membrane integrity loss. The PM-MIM approach, which is capable of demonstrating log removal value (LRV) above 4 for a fluorescent molecular marker (smaller in size than pathogens) should be suitable for providing a conservative estimate of pathogens LRV. However, regulatory acceptability of the present real-time PM-MIM approach will undoubtedly require long-term field evaluation in RO plants in order to establish the level of confidence to satisfy regulatory requirements for RO use in potable water reuse applications. (C) 2015 Elsevier B.V. All rights reserved.