As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.