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Thousands of unannotated small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been revealed in mammalian genomes. While hundreds of mammalian smORF- and alt-ORF-encoded proteins (SEPs and alt-proteins, respectively) affect cell proliferation, the overwhelming majority of smORFs and alt-ORFs remain uncharacterized at the molecular level. Complicating the task of identifying the biological roles of smORFs and alt-ORFs, the SEPs and alt-proteins that they encode exhibit limited sequence homology to protein domains of known function. Experimental techniques for the functionalization of these gene classes are therefore required. Approaches combining chemical labeling and quantitative proteomics have greatly advanced our ability to identify and characterize functional SEPs and alt-proteins in high throughput. In this review, we briefly describe the principles of proteomic discovery of SEPs and alt-proteins, then summarize how these technologies interface with chemical labeling for identification of SEPs and alt-proteins with specific properties, as well as in defining the interactome of SEPs and alt-proteins.