Atomic force microscopy (AFM) can be used to reveal intimate details about the effect of anesthetics on phospholipid bilayers. In AFM, surfaces are probed using a tip revealing lateral structural features at 10–20-nm resolution and height features at 0.5-nm resolution. Additionally, information on the viscoelasticity of the surface can be gained by examining the forces of tip-surface interactions. This is also known as force spectroscopy. In this chapter, the use of AFM to observe and quantify anesthetic-induced changes in phospholipid bilayers is detailed. The procedures developed to create supported phospholipid bilayers are described and the techniques developed to generate the best AFM images and force spectroscopy results have been revealed.