Membrane processes have revolutionized many industries because they are more energy and environmentally friendly than other separation techniques. This initial selection of the membrane for any application is based on its Molecular Weight Cut-Off (MWCO). However, there is a lack of a quantitative, liable, and rapid method to determine the MWCO of the membrane. In this study, a methodology to determine the MWCO, based on the retention of fluorescent silica nanoparticles (NPs), is presented. Optimized experimental conditions (Transmembrane pressure, filtration duration, suspension concentration, etc.) have been performed on different membranes MWCO. Filtrations with suspension of fluorescent NPs of different diameters 70, 100, 200 and 300 nm have been examined. The NPs sizes were selected to cover a wide range in order to study NPs diameters larger, close to, and smaller than the membrane pore size. A particle tracking analysis with a nanosight allows us to calculate the retention curves at all times. The retention rate curves were shifted over the filtration process at different times due to the fouling. The mechanism of fouling of the retained NPs explains the determined value of the MWCO. The reliability of this methodology, which presents a rapid quantitative way to determine the MWCO, is in good agreement with the value given by the manufacturer. In addition, this methodology gives access to the retention curve and makes it possible to determine the MWCO as a function of the desired retention rate.