Chinese hamster ovary (CHO) cells are ubiquitously used for therapeutic protein production. However, fed-batch culture, typically used for large-scale production, often induces hyperosmotic stress, negatively impacting cell growth and productivity. To identify genes conferring resistance to hyperosmotic stress, we performed genome-wide CRISPRa screening in bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells. Using a virus-free recombinase-mediated cassette exchange (RMCE) system, we established a CRISPRa library and cultured cells in standard and hyperosmolar media. Next-generation sequencing identified 122 significantly enriched and 171 significantly depleted genes under hyperosmolar conditions, with functional enrichment analysis highlighting pathways related to cell proliferation and transcriptional regulation. Among the enriched genes, CRISPRa-based activation of 24 candidates demonstrated that 23 improved cell growth under hyperosmolar conditions. Notably, stable expression of Siah2 or C2cd4a significantly enhanced cell growth, and optimizing their expression levels increased bsAb production by up to 1.3-fold. Additional knockout of Zfr, previously identified in CRISPR knockout screening, further improved cell growth and bsAb production, demonstrating the synergistic benefits of integrating CRISPR knockout and CRISPRa approaches. Thus, CRISPRa screening is a powerful tool for identifying novel engineering targets, facilitating the development of stress-resistant CHO cell lines, and enhancing therapeutic protein production.