Giant unilamellar vesicles (GUVs) have gained great popularity as mimicries for cellular membranes. As their sizes are comfortably above the optical resolution limit, and their lipid composition is easily controlled, they are ideal for quantitative light microscopic investigation of dynamic processes in and on membranes. However, reconstitution of functional proteins into the lumen or the GUV membrane itself has proven technically challenging. In recent years, a selection of techniques has been introduced that tremendously improve GUV-assay development and enable the precise investigation of protein–membrane interactions under well-controlled conditions. Moreover, due to these methodological advances, GUVs are considered important candidates as protocells in bottom-up synthetic biology. In this review, we discuss the state of the art of the most important vesicle production and protein encapsulation methods and highlight some key protein systems whose functional reconstitution has advanced the field.

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