Updated project metadata. Proteogenomic and ribosome profiling identification of translated small open reading frames have revealed thousands of microproteins, or polypeptides of less than 100 amino acids, that were previously invisible to geneticists. However, the vast majority of microproteins remain uncharacterized in molecular detail, in part because their lack of conservation and short lengths preclude analysis of homology to protein domains of known function. Proximity-dependent biotinylation technique provided an alternative approach to define the composition of subcellular compartments in living cells and animals. Here, we developed the first high-throughput technology for global mapping of cryptic microproteins to specific subcellular localizations and organelles by proximity-dependent biotinylation technology, thereby coupling their discovery to a dimension of biological functional information. In this work, we showed that hundreds of microproteins are subnuclear organelles associated with critical cellular and biological functions. Moreover, one of these novel microproteins, alt-LAMA3, localized in the nucleolus, is functionally associated with pre-rRNA transcription and required for global protein synthesis during ribosome biogenesis. Lastly, we applied this technique to mouse models, demonstrating the foundation for its use in discovery and characterization of microproteins in animals.