Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in??vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye PicoGreen. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo???, where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values.