Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.