Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution. A case study is reported to show the practicability of GV microfiltration. A clickable fluorescent probe was encapsulated inside GVs; more than 99.9% of the non-entrapped probe was easily and rapidly removed by multiple microfiltrations. This novel methodology is briefly discussed as a future tool for selection experiments on GV populations.