The intrinsic sensitivity (r 6 dependence) of F€ orster (or fluorescence) resonance energy transfer (FRET) to nanoscale changes in the donor/acceptor separation distance has made FRET an invaluable biophysical tool in a variety of applications, ranging from studying the structure and conformation of proteins and nucleic acids to examining biomolecular interactions, including its use in in vitro and in vivo bioassays [1][2][3][4][5][6][7]. While the myriad of FRET configurations and techniques currently in use are covered throughout this book, here we focus primarily on the materials utilized as donor or acceptor probes in FRET rather than the process itself [3,5,8,9]. Our 2006 review paper on this topic serves as the foundation for this updated chapter [3]. The materials were divided into three main categories: organic materials that include "traditional" dye fluorophores, dark quenchers, polymers, and carbon nanomaterials (NMs); inorganic materials such as metal chelates, metal, and semiconductor nanocrystals; and fluorophores of biological origin such as fluorescent proteins (FPs), amino acids, and fluorescence generated from enzymatic bioluminescence (BL) and chemiluminescence (CL). These materials may function as FRET donors and/or acceptors, depending upon experimental design. Many of the new materials developed and/or new donor-acceptor probe combinations used address some of the inherent complications of more traditional FRET materials, including photobleaching, spectral cross talk, and direct excitation of the acceptor species, and examples of these will be highlighted and discussed throughout the chapter. Since the vast majority of FRET applications are biological in nature, they routinely involve some type of biomolecule labeling strategy, which ultimately plays a significant and fundamental role in the success and interpretation of the resulting FRET. Therefore, we begin the chapter with a brief discussion of the bioconjugation techniques commonly utilized for FRET and points to consider, followed by sections highlighting the current and emerging materials that are used, or have the potential to be used, in FRET applications.