While, according to the World Health Organization, the successful application of antiretroviral therapy reduced global deaths from HIV/AIDS by 24% between 2005 and 2011, a corresponding rise in viral resistance to these drugs has been measured. This rise is of particular concern in low-resource settings that are collectively burdened with over two-thirds of worldwide HIV infections while lacking access to robust diagnostic assays. In response to this need, the Lutz Lab, in collaboration with the Frenkel Lab at Seattle Children’s Research Institute and the Lai Lab at UW BIOE, aims to engineer a low-cost paper-based oligonucleotide ligation assay (OLA) for drug-resistant HIV. In order to lower the chance of user errors and reduce diagnostic turnaround time, it is desirable to be able to detect a set of six point mutations indicative of drug-resistance to first-line antiretroviral treatment in a single device. However, with current techniques we are limited to being able to test for one point mutation per device. To address this challenge, we have investigated a DNA hybridization method of OLA probe capture. Using this method, each point mutation is assigned a specific DNA “barcode” that could be used to generate spatially distinct capture lines in a diagnostic device. We have designed a set of six engineered barcodes that display highly specific and sensitive binding to their engineered complements. Furthermore, the performance of this DNA barcode system has been compared to barcodes constructed from non-natural nucleic acid analogs in order to develop a method that optimizes sensitivity and specificity of probe capture. The completion of this project brings the OLA one step closer to being able to make a previously expensive, technology intensive, and operationally complicated test, accessible to those in low-resource settings.