<span class="paragraphSection"><div class="boxTitle">Abstract</div>The characterization of protein–DNA interactions underpinning fundamental biological processes requires methods that are laborious and time-consuming. Here, we introduce a rapid and instrument-free assay leveraging on GFP-tagged proteins and the lateral flow assay principle to examine protein–DNA interactions. The rapid protein–DNA interaction test (R-PNAI-T) detects complexes using a dipstick in ∼15 min. Validation of the R-PNAI-T with bacterial proteins involved in replication and transcription demonstrated its applicability for diverse protein–DNA interactions, achieving a remarkable detection sensitivity of ∼0.7 fmol with the <span style="font-style: italic;">Escherichia coli</span> Tus protein. Analysis of these protein interactions with their specific target DNA sequences highlighted the capability of the R-PNAI-T to discern subtle differences in affinity, providing valuable comparative data. The R-PNAI-T is robust, retaining functionality in human serum and bacterial lysates. The assay showed tolerance towards biotin contaminants, expanding its use for quality control in protein purification processes and tracking complexes in biological samples. To demonstrate versatility, we applied the R-PNAI-T with polymerase chain reaction-amplified DNA to probe the putative origin of replication in <span style="font-style: italic;">Burkholderia pseudomallei</span>, confirming a functional interaction between the DnaA initiator protein and the DnaA box in this bacterium. Overall, the R-PNAI-T is versatile, cost-effective, and user-friendly, offering broad applications in biological research and biotechnology.</span>