Several amphipathic compounds are able to interact with biological membranes and perturb their structure or disrupt their integrity. In particular, detergents are routinely used to solubilize and separate membrane components and antimicrobial peptides are able to kill pathogens by disrupting their membrane. The mechanism of action of such membrane active agents can be described and elucidated using giant unilamellar vesicles (GUVs), because they are cell-sized objects that allow for direct visualization. In this chapter, several approaches using optical microscopy of GUVs relevant for the study of the mode of action of antimicrobial peptides and the detergent solubilization process are presented. In special, experimental protocols to quantify membrane permeability and increase in membrane area are shown in detail, and ways to assess the types of membrane perturbations induced by membrane-active agents are discussed.