The authors present a novel technique for real-time visualization and measurement of DNA replication by individual replisomes. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, they monitor the progression of single-DNA synthesis events and measure rates and processivities of T7 and Escherichia coli replisomes. This method allows for rapid and precise characterization of DNA synthesis kinetics and the effects of replication inhibitors. The rolling-circle replication scheme facilitates highly processive DNA synthesis, and the technique is applicable to both bacterial and viral DNA replication systems. The authors demonstrate the assay's effectiveness by characterizing fully reconstituted replisomes from T7 and E. coli, showing rates of 75.9 ± 4.8 bp/s and 535.5 ± 39 bp/s, respectively, with average processivities of 25.3 ± 1.7 kbp and 85.3 ± 6.1 kbp. The assay is also used to study the effects of dideoxynucleotide triphosphate (ddGTP) on replication rates and processivity, providing a powerful tool for screening and characterizing replication inhibitors.The authors present a novel technique for real-time visualization and measurement of DNA replication by individual replisomes. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, they monitor the progression of single-DNA synthesis events and measure rates and processivities of T7 and Escherichia coli replisomes. This method allows for rapid and precise characterization of DNA synthesis kinetics and the effects of replication inhibitors. The rolling-circle replication scheme facilitates highly processive DNA synthesis, and the technique is applicable to both bacterial and viral DNA replication systems. The authors demonstrate the assay's effectiveness by characterizing fully reconstituted replisomes from T7 and E. coli, showing rates of 75.9 ± 4.8 bp/s and 535.5 ± 39 bp/s, respectively, with average processivities of 25.3 ± 1.7 kbp and 85.3 ± 6.1 kbp. The assay is also used to study the effects of dideoxynucleotide triphosphate (ddGTP) on replication rates and processivity, providing a powerful tool for screening and characterizing replication inhibitors.