Abstract: Advanced methods in next-generation sequencing and proteogenomics have revealed thousands of previously invisible human protein-coding genes, increasing the known size of the human proteome by at least 10%. This previously unannotated proteomic “dark matter” includes small open reading frames (smORFs) encoding polypeptides of fewer than 100 amino acids, and alternative open reading frames (alt-ORFs) encoding proteins 100 amino acids or larger. Sm/alt-ORFs previously escaped detection due to their short lengths, overlap with annotated protein coding sequences in different reading frames, and/or initiation with non-AUG start codons. Recent studies have shown that hundreds of smORFs are required for cell growth and survival, and some smORF-encoded polypeptides or “microproteins” bind to and regulate the activity of macromolecular complexes involved in critical cellular processes and disease. However, the functions and phenotypes associated with smORFs and alt-ORFs remain almost entirely unexplored. We describe chemical and quantitative proteomic strategies to profile sm/alt-ORF-encoded microproteins that exhibit properties of interest, such as post-translational modifications, regulated translation and subcellular localization. Using these tools, we identified and mechanistically characterized human microproteins that regulate RNA granule formation, inositide signaling and ribosome biogenesis. Additionally, I will present unpublished data showing that two novel proteins control epigenetic processes. Chemical and quantitative proteomic strategies may therefore be leveraged to accelerate the identification of sm/alt-ORFs with significant biological functions in order to obtain new insights into human cell biology.