DNA polymerase theta (Pol θ) is involved in an end-joining pathway of DNA double strand breaks. Over expression of Pol θ is found in many cancers, including stomach, colon, breast and lung cancers, and correlated with poorer patient survival. Because suppression of gene expression of Pol θ results in sensitivity of cells to ionizing radiation and some DSB-inducing drugs, Pol θ is a validated anti-cancer drug target. The assay is fast, convenient, and requires just two steps. In the first step, the DNA Pol θ enzyme synthesizes double-stranded DNA using a DNA template in the presence of dNTP. In the second step, a dye that binds to double-stranded DNA is added to the solution resulting in the increase of fluorescence, intensity of which can be measured with a fluorescent plate reader at the excitation wavelengths of 495 nm and emission wavelengths of 525 nm.