The biomimetic membrane technology may unlock unprecedented membrane separation capabilities to solve the increasing need for clean water. Despite the efforts in exploring numerous membrane preparation methods, the membrane performance achieved to date is still far from the theoretical predictions. To overcome this bottleneck, a deeper understanding of the role of the channels or vesicles immobilized on the membrane would be required. In this work, we seek to quantify the amount of vesicles immobilized per unit area of membrane and correlate it with the membrane performance. The results show that, although the vesicles successfully immobilized onto the membrane increase with an increasing vesicle concentration, less than 4% of the vesicles loaded onto the membrane successfully remains on the membrane after interfacial polymerization. Furthermore, an increase in the amount of vesicles remaining on the membrane may not always result in improvement in membrane performance. To the best of our knowledge, this is the first time that a study has been performed to determine an accurate relationship between the vesicles immobilized and the biomimetic membrane performance.