The ever-increasing availability of genome sequencing data has revealed a substantial number of uncharacterized genes without known functions across various organisms. The first comprehensive genome sequencing of E. coli K12 revealed that more than 50% of its open reading frames corresponded to transcripts with no known functions. The group of protein-coding genes without a functional description and/or a recognized pathway, beginning with the letter “Y”, is classified as the “y-ome”. Several efforts have been made to elucidate the functions of these genes and to recognize their role in biological processes. This review provides a brief update on various strategies employed when studying the y-ome, such as high-throughput experimental approaches, comparative omics, metabolic engineering, gene expression analysis, and data integration techniques. Additionally, we highlight recent advancements in functional annotation methods, including the use of machine learning, network analysis, and functional genomics approaches. Novel approaches are required to produce more precise functional annotations across the genome to reduce the number of genes with unknown functions. Graphical abstract: [Figure not available: see fulltext.].