Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is a fluorescence based technique which enables the analysis of molecular interactions in biochemical processes. Principle of TR-FRET is based on time-resolved fluorescence (TRF) measurement and fluorescence resonance energy transfer (FRET) between donor and acceptor molecules. To generate FRET signal, donor and acceptor molecules must show spectral overlap and should be in close proximity to each other and display suitable dipole orientation. The specific signal is acquired from molecules of interest via interactions of donor and acceptor molecules. TR-FRET technique is widely used for studying kinase assays, cellular signaling pathways, protein-protein interactions, DNA-protein interactions, and receptor-ligand binding. There are various propriety applications of TR-FRET. Two different sample protocols are summarized in this review.