The characterization of membrane properties with and without overproduced proteins can be the first step in determining protein specific mechanism of action in a microorganism. However, not all proteins are easily purified and solubilized; addition to a model membrane for investigation is therefore often challenging. Even if the protein can be purified with ease, crucial elements of the mechanism of action may remain hidden in an overly simplified experiment employing model membranes. We propose a more elegant approach that considers time-resolved fluorescence measurements using inner-membrane vesicles (INV) as an in vitro model without prior protein purification.