A high-quality genome annotation greatly facilitates successful cell line engineering. Standard draft genome annotation pipelines are based largely on de novo gene prediction, homology, and RNA-Seq data. However, draft annotations can suffer from incorrect predictions of translated sequence, inaccurate splice isoforms and missing genes. Here we generated a draft annotation for the newly assembled Chinese hamster genome and used RNA-Seq, proteomics, and Ribo-Seq to experimentally annotate the genome. We identified 3,529 new proteins compared to the hamster RefSeq protein annotation and 2,256 novel translational events (e.g., alternative splices, mutations, novel splices). Finally, we used this pipeline to identify the source of translated retroviruses contaminating recombinant products from Chinese hamster ovary (CHO) cell lines, including 119 type-C retroviruses, thus enabling future efforts to eliminate retroviruses by reducing the costs incurred with retroviral particle clearance. In summary, the improved annotation provides a more *