The oxysterol-binding protein/OSBP-related proteins (OSBP/ORPs) are a family of proteins conserved in all eukaryotes that have complex biological activities connected to lipid transport and lipid regulation. OSBP is ubiquitously expressed in tissues and is required in the replication of a broad array of pathogenic Enterovirus species. Alternatively, ORP4L is only expressed in a few select tissues and plays an important role in the proliferation and viability of certain cancers. Although the OSBP/ORPs have been reported to interact with an array of ligands, including various sterols, phospholipid compounds, and natural product compounds, the comprehensive characterization of ligand binding to the OSBP/ORP proteins has not been performed. Additionally, this is the first binding study on human OSBP and ORP4L, an important step for designing ligands for therapeutic targeting of the ORPs. The goal of this research is to characterize the ligand binding of human OSBP and ORP4. We utilized two experimental approaches to understand the small molecule ligand binding ability of OSBP and ORP4L. The first experimental approach was to determine binding affinities of multiple classes of potential ligands for OSBP and ORP4L, which was accomplished through the implementation of a high-throughput ligand binding assay using cloned and expressed human OSBP and ORP4L proteins. Through screening oxysterols for ligand binding, specific sites of oxysterol side chain oxidation were identified as being critical for high-affinity interaction with OSBP and ORP4. Specifically, oxysterols that show high-affinity binding with OSBP and ORP4L have hydroxyls at the C20, C24, C25, C26 or C27 positions, but not at the C22. The importance of the side chain in oxysterol binding was further determined by testing a series of 20-hydroxycholesterol analogs produced in our lab. The second experimental approach employed was to construct and test a structural model of how OSBP interacts with its small molecule ligands, specifically the natural product compound OSW-1. Using the existing partial OSBP/ORP structures from yeast orthologs, we constructed a model for OSW-1-OSBP interactions. The tentative model was used to identify OSBP potentially critical residues that are essential for OSW-1 binding, and the identified amino acids were selected for mutation. One OSBP point mutant was successfully cloned, expressed and tested for ligand binding. The OSBP H522A mutant negatively affects OSW-1 binding while not affecting oxysterol binding, which supports our interaction model. The results of these projects inform us of how small molecule ligands bind OSBP and ORP4L, and perhaps by analogy the other OSBP/ORPs. These research accomplishments will aid in the design of new generations of OSBP/ORP binding small molecules, including potentially novel anti-cancer and anti-viral compounds for therapeutic development to selectively target only OSBP or only ORP4.