Ca2+ signals control a vast array of cellular processes and are mediated by the concerted effort of a spectrum of Ca2+ channels, transporters, and pumps present in the plasma membrane (PM) and endoplasmic reticulum (ER) membrane [1,2]. In nonexcitable cell types, store-operated channels (SOCs) are the major means through which extracellular Ca2+ enters cells to generate Ca2+ signals. The two major components of SOCs, STIM1 and Orai1, were identified a decade ago [3–8], and extensive studies have focused on the mechanisms of how STIM1 becomes activated in response to store depletion, how Orai1 subunits assemble to form the channel, and how the STIM1 molecule interacts with Orai1 to achieve channel gating [1,9–13] (discussed in Chapters 2 and 3). The physical interaction between STIM1 and Orai1 has been one of the most important parameters in order to understand the stoichiometry and gating mechanism of the STIM1/Orai1 complex. The physical interaction between STIM and Orai1 has been studied extensively using Forster (fluorescence) resonance energy transfer (FRET) imaging technology [13–16]. FRET allows the visualization and quantification of macromolecular interactions in living cells by measuring light energy transfer between closely associated fluorescently tagged proteins [17]. Since STIM1 and Orai1 are in different membranes and interact at discrete ER-PM junctions, FRET is a highly effective means for assessing this interaction. FRET occurs in a short range (5–10 nm) across which energy is transferred from an excited donor to acceptor fluorophore [18]. The efficiency of energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor fluorophores; hence, FRET measurements give extremely sensitive information on the distance separating the pair. The mechanism and uses of FRET are well described in other reviews [19–23].Despite intense study, the molecular nature of the coupling interaction between the activated STIM1 protein and the Orai1 channel remains elusive. Our approach to studying this interaction is to use a fragment of the STIM1 protein that itself is able to mediate full activation of the Orai1 channels. This 100-amino acid fragment is known as SOAR (STIM-Orai-activating region) [24,25] (see Chapter 2). In a previous report, Shen et al. [26] purified and crystallized this fragment from STIM1 and revealed that it can exist as a dimer. Indeed, the SOAR fragment appears to be an important “core” structure within the STIM1 protein contributing to dimerization of the whole STIM1 protein [12]. We have been able to express the SOAR protein as a concatenated dimer construct and therefore genetically manipulate the exact dimeric composition of SOAR expressed in cells [13]. We revealed that a single point mutation (F394H) in the Orai1 binding site of the SOAR fragment from STIM1 can completely prevent STIM1 binding to and activation of Orai1 channels [16]. Importantly, since the SOAR dimer contains two of these sites, we can modify either one or both of these sites to study the requirements for interaction with the Orai1 channel. Using a set of concatemer-dimers of SOAR containing one or two F394H mutations, we are able to study how the SOAR dimer interacts with Orai1 and whether each SOAR unit within a dimer is equivalent. The aim of this chapter is to provide a detailed protocol to assess the STIM/Orai interaction by FRET. We describe how to ensure the FRET assay is reliable and consistent and how to analyze and how to interpret the FRET data.