A simple technique for real-time visualization of individual DNA molecule replication is presented. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, the progression of single-DNA synthesis events is monitored, allowing accurate measurement of replication rates and processivities of T7 and Escherichia coli replisomes. This method enables rapid and precise characterization of DNA synthesis kinetics and the effects of replication inhibitors. DNA replication involves a complex interplay of enzymes organized into a replisome. Traditional biochemical methods rely on agarose gel electrophoresis and radioactive nucleotides, but these techniques are time-consuming and provide averaged data. Single-molecule techniques offer insights into replication dynamics, including short-lived intermediates. Recent studies have used flow-stretching techniques to observe enzyme-DNA interactions, but these methods provide indirect readouts of replication fork dynamics. The rolling-circle DNA amplification method is used to study coordinated DNA replication by individual replisomes in real time. The 5'-end of the lagging strand of a rolling-circle substrate is attached to the flow chamber surface, and replication components are introduced to initiate DNA synthesis. Hydrodynamic flow stretches the growing DNA, and a low concentration of intercalating stain allows real-time imaging of DNA length. This technique characterizes fully reconstituted replisomes from T7 and Escherichia coli systems, which are large protein complexes studied at the single-molecule level. The study demonstrates that T7 replication occurs at 75.9 ± 4.8 bp/s with an average processivity of 25.3 ± 1.7 kbp, while E. coli replication occurs at 535.5 ± 39 bp/s with an average processivity of 85.3 ± 6.1 kbp. The results show that E. coli replisomes have higher rates and processivities compared to T7 replisomes. The effects of dideoxynucleotides on replication rates and processivities are also examined, showing a decrease in both with increasing concentrations of dideoxynucleotides. The method allows for the real-time observation of DNA replication and the effects of replication inhibitors. The simplicity of the technique, combined with the ability to observe single active replisomes, makes it a powerful tool for studying DNA replication. The assay is suitable for high-throughput screening of replication inhibitors and other biochemical perturbations. The results demonstrate the potential of this method for studying the dynamics of DNA replication and the effects of various factors on replisome activity.A simple technique for real-time visualization of individual DNA molecule replication is presented. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, the progression of single-DNA synthesis events is monitored, allowing accurate measurement of replication rates and processivities of T7 and Escherichia coli replisomes. This method enables rapid and precise characterization of DNA synthesis kinetics and the effects of replication inhibitors. DNA replication involves a complex interplay of enzymes organized into a replisome. Traditional biochemical methods rely on agarose gel electrophoresis and radioactive nucleotides, but these techniques are time-consuming and provide averaged data. Single-molecule techniques offer insights into replication dynamics, including short-lived intermediates. Recent studies have used flow-stretching techniques to observe enzyme-DNA interactions, but these methods provide indirect readouts of replication fork dynamics. The rolling-circle DNA amplification method is used to study coordinated DNA replication by individual replisomes in real time. The 5'-end of the lagging strand of a rolling-circle substrate is attached to the flow chamber surface, and replication components are introduced to initiate DNA synthesis. Hydrodynamic flow stretches the growing DNA, and a low concentration of intercalating stain allows real-time imaging of DNA length. This technique characterizes fully reconstituted replisomes from T7 and Escherichia coli systems, which are large protein complexes studied at the single-molecule level. The study demonstrates that T7 replication occurs at 75.9 ± 4.8 bp/s with an average processivity of 25.3 ± 1.7 kbp, while E. coli replication occurs at 535.5 ± 39 bp/s with an average processivity of 85.3 ± 6.1 kbp. The results show that E. coli replisomes have higher rates and processivities compared to T7 replisomes. The effects of dideoxynucleotides on replication rates and processivities are also examined, showing a decrease in both with increasing concentrations of dideoxynucleotides. The method allows for the real-time observation of DNA replication and the effects of replication inhibitors. The simplicity of the technique, combined with the ability to observe single active replisomes, makes it a powerful tool for studying DNA replication. The assay is suitable for high-throughput screening of replication inhibitors and other biochemical perturbations. The results demonstrate the potential of this method for studying the dynamics of DNA replication and the effects of various factors on replisome activity.