Bottom-up synthetic biology aims to create biomimetic systems from the basic elements of cellular life. Such synthetic cell analogs may support scientists in understanding the fundamental biological behaviors at the cellular level. As microscopic aqueous compartments confined within a lipid bilayer, liposomes have been widely used as simplistic cellular mimics for synthetic cell studies. Here, we describe the manufacturing protocol for octanol-assisted liposome assembly (OLA) to produce unilamellar, monodispersed, cell-sized, and biocompatible liposomes as well as in situ liposome experiments. We use maskless, direct-write lithography, which allows for rapid prototyping, to fabricate PDMS-based lab-on-a-chip devices. OLA makes use of the double emulsion generation and dewetting purification approach, with the membrane composition of the formed liposomes and the nature of the encapsulated solution widely adjustable. Using highly responsive pressure-driven flow, the liposome size is tuneable with a steady liposome generation, and the experiment requires very low sample volumes (∼50 µl). We enrich the liposomes from the waste products using density-based on-chip separation techniques, which allows us to conduct experiments on the same device with real-time observation, right after production. As a proof-of-principle synthetic biology application, we demonstrate the capability of adjusting the microenvironment by inducing pH-controlled coacervation between ATP and poly-Llysine molecules inside the liposomes via transmembrane proton flux. Such liquid-liquid phase separation, creating multiple distinct phases from a single homogeneous mixture, is known to play a key role in a variety of cellular processes, including the formation of membraneless organelles as well as numerous other supramolecular assemblies. Our technique shows promising potential for synthetic cell research, particularly in terms of step-by-step construction and experimental control.