RIBO-seq and proteomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (sm/alt-ORFs <150 codons) that are translated into microproteins and alternative proteins (micro/alt-proteins). Several dozens of micro/alt-proteins have been characterized at the molecular level and demonstrated to play important biological roles. However, the overwhelming majority of micro/alt-proteins remain undefined, possibly because they are excluded from protein databases and cannot be detected by a classical proteomic workflow. Here, we introduce a proteomic pipeline to detect unannotated micro/alt-proteins in cultured cells or tissues from total protein extraction, gel-based size selection, digestion, and liquid chromatography-mass spectrometry proteomics (LC-MS/MS), to data analysis and manual validation. This approach is able to identify unannotated micro/alt-proteins from cultured cells and (patho)physiological tissues.