Peripheral blood samples from patients diagnosed as HIV-1 positive and treated with anti-retroviral therapy (ART) for prolonged periods of time can present difficulties in current quantitative HIV-1 DNA detection, based on PCR assays. The current gold standard platform for HIV-1 DNA detection is real-time PCR, but its sensitivity is not always enough to detect extremely low levels of HIV-1 DNA in some patients. We have developed a novel end-point PCR assay based on precision image pi-code (πCode) MicroDiscs detection platform. The newly developed "πCode assay" showed 100% sensitivity, while the real-time PCR assay showed 92.3%, in a head-to-head comparison using blood samples from 39 HIV-1 infected clinic patients, with a stable fully suppressed plasma viral-load (<20 copies/mL) for more than 2 years. Further analysis revealed a detection sensitivity over 27 times higher for the "πCode assay" compared to real-time PCR. The πCode assay is very specific and reproducible, with less than 20% Coefficient of Variation (CV) in detection, ranging from 40 to 4000 HIV-1 DNA copies per one million white blood cells. The πCode assay may have a great potential to be a standard assay platform in the future for the HIV diagnostic field, focusing on HIV cure trials.