Protein-nucleic acid interactions underpin fundamental biological processes, furthering the continual development of methods enabling their study. Traditional methods are often laborious and time-consuming. Here, we introduce a rapid and instrument-free assay leveraging on the lateral flow assay principle and GFP-tagged proteins. The rapid protein-nucleic acid interaction test (R-PNAI-T) captures and detects protein-DNA or protein-RNA complexes using a commercially available dipstick, yielding results in less than 30 minutes. Validation of the R-PNAI-T demonstrated its robustness and reliability across diverse DNA-binding proteins, achieving a remarkable detection sensitivity of ~15 pM. Analysis of protein interactions with canonical DNA sequences highlighted the capability of the R-PNAI-T to discern subtle differences in binding affinities, providing valuable data for comparing PNAIs. The R-PNAI-T is robust demonstrating resilience to biological matrix effects, retaining functionality in complex samples like human serum, with only minor analytical sensitivity reduction observed in bacterial lysates. Moreover, the assay showed tolerance towards biotin contaminants, further expanding its potential applications for quality control in protein purification processes and for tracking protein-nucleic acid complexes in biological samples. To demonstrate versatility, we applied the R-PNAI-T to probe the putative origin of replication in Burkholderia pseudomallei, confirming for the first time a functional interaction between the DnaA initiator protein and the DnaA box in this bacterium. Overall, the R-PNAI-T is a rapid, versatile, cost-effective, and user-friendly assay for studying PNAIs, with broad applications in biological research and biotechnology.