<p>Elastin-like polypeptides (ELPs), composed of repeating pentapeptide units, are thermo-responsive, protein-based biopolymers that undergo phase transition. The expression levels of ELPs are known to vary depending on the identity of the guest residue (<i>X</i>) and are significantly reduced in longer ELP sequences. This phenomenon is hypothesized to be influenced by differences in codon composition and the limited availability of specific aminoacyl-tRNAs corresponding to Val, Pro, Gly, and <i>X</i> residues. In this study, we prepared green fluorescent protein (GFP)-fused ELPs of varying lengths, guest residue compositions, and codon sequences to systematically evaluate their expression levels. Fluorescence intensity of GFP was used as a rapid and quantitative measure of ELP expression. To investigate the underlying causes of reduced expression in long ELPs, we analyzed the codon usage, identified the presence of rare codons, and evaluated strategies to mitigate their impact. Supplementation of cells with tRNAs for rare codons and codon sequence optimization were explored as potential solutions. Notably, the expression level of a 128-mer ELP was enhanced by approximately ninefold with tRNA supplementation. This study presents a robust approach for the quantitative analysis of repetitive polypeptide expression and offers an effective strategy to improve expression yields. These findings have significant implications for the scalable production of repetitive protein-based biopolymers in research and industrial applications.</p>