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Summary: Cyclic-phospholipids-based vesicles can play a role in facilitating the chemical evolution of protocells from the structurally simple to the functionally more complex form. Here, we present a protocol for preparing decanoic acid-derived cyclic phospholipid and glyceryl-diester phosphate-containing vesicles. We describe steps for sample preparation, equilibration, and image acquisition using confocal microscopy. This protocol has the potential for preparing a wide variety of these phospholipid-based artificial cell constructs.For complete details on the use and execution of this protocol, please refer to Pulletikurti et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.