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Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. KEY MESSAGES: Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.