Fluorescence excitation-emission matrix (EEM) spectroscopy was used to distinguish between two stages of reverse osmosis (RO) permeates as the first step towards investigating the potential application of fluorescence as a monitoring tool for membrane performance. The signal response of several fluorescence peaks present in Stage 1 and Stage 2 RO permeates of an advanced water treatment plant were compared. The humic-like fluorescence region was found to have the largest percentage difference between stages and therefore was the most appropriate for enabling differentiation. Increases in humic-like fluorescence did not correlate with increases in conductivity or dissolved organic carbon measurements. This suggests that fluorescence is a more selective and sensitive method for monitoring the organic composition of RO permeates than established methods. Fluorescence is therefore a promising tool for improved water quality monitoring of RO permeates.