The result of nonsense mutation is premature stop-codon (PTC) in an open reading frame of a gene, resulting in truncated, inactive protein product. This leads to many diseases like cystic fibrosis, Duchenne muscular dystrophy, cancers and others. Small molecules called nonsense suppressors (NonSups) can stimulate stop-codon readthrough, inducing the ribosome to accept a near-cognate tRNA at the PTC, and restoring length and function of the protein. To elucidate the mode of action of NonSups, we have developed a pathway-specific, in vitro eukaryotic readthrough assay. Internal ribosome entry site containing mRNA from cricket-paralysis virus (CrPV-IRES) was chosen to by-pass the need for eukaryotic translation initiation factors. To understand the cap-independent translation initiation by CrPV-IRES, I measured the pseudo-translocation of CrPV-IRES and the translocation of CrPV-IRES and the first coding tRNA using fluorescence anisotropy and tRNA co-sedimentation, and translocation of the third and fourth elongation cycle using puromycin reaction with peptidyl P-site tRNA. I have shown that it is possible to produce at least a tetrapeptide using CrPV-IRES, with the caveat that the rate of translation initiation is slowed by two pseudo-translocation steps and two subsequent genuine translocation steps. To observe NonSup-stimulated PTC readthrough, I employed CrPV-IRES in a highly purified cell-free eukaryotic translational assay. Of the 16 NonSups tested, 12 exhibited readthrough activity in this assay of translational machinery activity. A closer inspection of the readthrough behaviors revealed at least two possible distinctive stop-codon readthrough mechanisms by aminoglycosides and ataluren-like molecules. To investigate the mechanism of NonSup-induced readthrough in detail, I studied three steps of translation elongation during PTC readthrough: tRNA association to the ribosome and translocation, measured by fluorescence spectroscopy, and peptide bond formation determined using thin layer electrophoresis purification of the readthrough peptide product. I found that G418, chosen as an example of an aminoglycoside, induces PTC readthrough by stimulating faster peptide bond formation. In contrast, ataluren induces PTC readthrough by inhibiting peptide release catalyzed by release factors eRF1 and eRF3. These findings will inform medicinal chemists in their utilization of rational drug design to create enhanced and safer NonSups as treatments for patients with diseases caused by nonsense mutations.