ABSTRACT Understanding the information contained within the human genome has been a goal of molecular biology and medicine since the identification of DNA. The Human Genome Project cataloged all the protein-coding genes embedded within the genome, but gene-finding algorithms had a blind spot for protein-coding small open reading frames (smORFs). Technological advances over the last decade have led to the discovery of thousands of smORFs in the human genome that encode peptides or small proteins (median length 30 amino acids) collectively referred to as microproteins. The discovery of thousands of previously unknown proteins in the human genome is an exciting opportunity in molecular biology that promises to improve scientific understanding and the ability to discover and develop new medicines. This proposal seeks to answer important questions that have emerged regarding microproteins, including whether microproteins from polycistronic genes are functional (Aim 1), whether biologically active secreted microproteins exist (Aim 2), and, lastly, whether it is possible to use knowledge about the structure and mechanism of microproteins to develop small-molecule probes that can regulate their actives in cells (Aim 3). These questions will be tackled using approaches and reagents developed over the last decade. For example, optimized robust protocols are now available for investigating microprotein-protein interactions together with necessary reagents including antibodies, cell lines, and genetically engineered mice to study these unique genes. This proposal will reveal new functions for microproteins in cells and tissues, identify biologically active secreted microproteins, and identify small molecule probes of microprotein biology. These efforts will increase understanding of the roles of smORFs and microproteins in mammalian genomes and identify secreted microproteins and small molecules that provide a template for converting these foundational discoveries into medicines.