Digital PCR is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides a high flexibility in assay design without influencing quantitative accuracy. Here, an assay is described to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence.