Abstract Drug resistance to antiretroviral therapy for HIV infection is a serious clinical problem without cost-effective solutions in low and middle-income countries (LMICs) where standard genotypic resistance assays are unaffordable. Discidium's goal is to develop a single tube assay that uses lyophilized reactants, standard PCR and in-tube readout, that can be used as a screening resistance test and in laboratories with minimal infrastructure. We have exploited a Taq polymerase that absolutely requires a terminal 3´ base match that, with appropriate primers matched to resistance-associated polymorphisms, forms the basis for a low-cost allele- specific PCR resistance assay. This assay reveals the presence of first line resistance mutations to the most common NRTI that underscore resistance in the vast majority of individuals failing therapy in LMICs. We have also developed a fluorescence approach that allows direct visualization of PCR products under a blue light source. As such, the assay is single-step in that amplification and readout occur in the same tube. Our preliminary data supports the use of this assay to target the most common mutations that confer resistance to ARTs (K103N for NNRTIs and M184V for NRTIs), and we propose to optimize the use of this assay in unprocessed blood, and rigorously validate the assay performance using HIV-1 harboring the K103N and M184V mutations. This assay has the potential to be developed into a complete kit that health care workers can use to perform HIV-1 resistance testing in LMICs to guide optimal management of HIV-1-infected individuals.