LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins are important mediators of cell specification, proliferation and differentiation. These proteins all contain two tandem LIM domains that mediate interactions with other proteins, including LIM domain binding protein 1 (LDB1), an important co-factor that facilitates their roles in development and disease. As all 16 mammalian LMO and LIM-HD proteins compete for binding to LDB1, we want to establish the relative binding affinities for this series of interactions. The LIM domains for these proteins tend to be insoluble and prone to aggregation, making this difficult by standard interaction methods.We have designed a Förster Resonance Energy Transfer (FRET)-based competition binding assay to study LIM:LDB1 interactions. The sensitivity of FRET to distances of 1-10 nm means it is a useful tool in studying protein-protein interactions. We have designed and produced a fusion construct containing the LIM domains of LMO4 and the LIM interacting domain (LID) of LDB1 with a fluorescent protein pair optimised for FRET experiments¹ . Preliminary data from the assay suggests that LMO4 binds LDB1 with a low nanomolar affinity, consistent with previous ELISA studies. This has been further validated using a fluorescence gel retardation competition assay. With further optimisation this system may be a simple and powerful tool for studying LIM:LDB1 interactions in solution.