Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing regulated synthesis could play important cellular roles, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated alt-proteins or N-terminal extensions, one of which is post-transcriptionally upregulated by DNA damage stress. We further defined cell cycle-regulated MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar alt-protein co-encoded with human RBM10. Depletion of MINAS-60 increases the amount of cytoplasmic 60S ribosomal subunit, upregulating global protein synthesis and cell proliferation. Mechanistically, MINAS-60 represses the rate of late-stage pre-60S assembly and export to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of pre-60S maturation, and demonstrate that chemoproteomics can enable functional hypothesis generation for uncharacterized alt-proteins.