Protein–protein interactions regulate biological networks. The most proximal events that initiate signal transduction frequently are receptor dimerization or conformational changes in receptor complexes. Toll-like receptors (TLRs) are transmembrane receptors that are activated by a number of exogenous and endogenous ligands. Most TLRs can respond to multiple ligands and the different TLRs recognize structurally diverse molecules ranging from proteins, sugars, lipids, and nucleic acids. TLRs can be expressed on the plasma membrane or in endosomal compartments, and ligand recognition thus proceeds in different microenvironments. Not surprisingly, distinctive mechanisms of TLR receptor activation have evolved. A detailed understanding of the mechanisms of TLR activation is important for the development of novel synthetic TLR activators or pharmacological inhibitors of TLRs. Confocal laser scanning microscopy (LSM) combined with green fluorescent protein (GFP) technology allows the direct visualization of TLR expression in living cells. Fluorescence resonance energy transfer (FRET) measurements between two differentially tagged proteins permit the study of TLR interaction and distances between receptors in the range of molecular interactions can be measured and visualized. Additionally, FRET measurements combined with confocal microscopy provide detailed information about molecular interactions in different subcellular localizations. Bimolecular complementation using split fluorescent proteins (BiFC) represents an additional valuable method to study mechanisms of receptor activation in living cells. These techniques permit the dynamic visualization of early signaling events in living cells and can be utilized in pharmacological or genetic screens.