The quality control of RNA has become increasingly crucial with the rise of mRNA-based vaccines and therapeutics. However, conventional methods such as LC-MS often require specialized equipment and expertise, limiting their applicability to high throughput experiments. Here, we optimize a previously characterized RNA integrity biosensor, that provides a simple colorimetric output, using Design of Experiments (DoE). Through iterative rounds of a Definitive Screening Design (DSD) and experimental validation, we systematically explored different assay conditions to enhance the biosensor's performance. Optimization led to a 4.1-fold increase in dynamic range and reduced RNA concentration requirements by one-third, significantly improving usability. Notable modifications included reducing the concentrations of reporter protein and poly-dT oligonucleotide and increasing DTT concentration, suggesting a reducing environment for optimal functionality. Importantly, the optimized biosensor retained its ability to discriminate between capped and uncapped RNA even at lower RNA concentrations. Overall, our improved biosensor offers enhanced performance and reduced sample requirements, paving the way for rapid, cost-effective RNA quality control in diverse settings, including resource-limited environments.