Publisher Summary This chapter discusses methods that can be used to measure the pore-forming activity of type III secretion systems (TTSSs) in host eukaryotic cells. The process by which TTSS toxins are delivered across host membranes is not well understood. Some TTSSs can introduce pores into plasma membranes of host eukaryotic cells or synthetic lipid bilayers. Pore formation by a TTSS is generally dependent on the same proteins that are essential for toxin translocation. A current model suggests that TTSS toxins move through a transmembrane channel formed by components of the TTSS, and that pore formation by a TTSS results from the opening of translocation channels to the extracellular environment. Assays that measure pore formation using semipurified or purified TTSS components and synthetic membranes are also described. Many important issues remain to be addressed in this area. One of the most important issues is the source of the pores introduced by TFSSs. The methods described in the chapter may also be applied to the discovery of pore-forming activities associated with other types of bacterial secretion systems.