Mobility of proteins is crucial for their functionality. Fluorescence correlation spectroscopy (FCS) is a sensitive tool for assessing dynamics in vivo. It can reveal properties of diffusing proteins, as well as of the surrounding medium. Hence, subtle changes in the dynamics after treatment with toxic substances can be visualized. On biological membranes, the high concentration of transmembrane and peripheral membrane proteins leads to molecular crowding, and thus to a change in the diffusion behavior, i.e., to anomalous diffusion of membrane proteins. Presented here is a protocol for conducting and evaluating FCS measurements of membrane proteins before and after treatment. Curr. Protoc. Toxicol. 48:2.19.1‐2.19.16. © 2011 by John Wiley & Sons, Inc.