Development of a novel host cell DNA assay using PicoGreen is described, which is capable of detecting short double stranded DNAs (ds-DNAs) in cell culture supernatants and process intermediates. Examination of this PicoGreen DNA assay was carried out by determination of the DNA length detection limit, observation of short ds-DNAs in cell culture supernatants and process intermediates, evaluation of dose dependency and a supersensitizing protocol, and comparison of the novel assay with conventional assays for measuring host cell DNA concentration in real samples. The PicoGreen DNA assay was capable of detecting ds-DNAs as short as 20 bp, and the sensitivity of the PicoGreen DNA assay was comparable to that of the Threshold system with application of additional SDS/Proteinase K digesting and DNA concentrating steps. Also, the amount of DNA identified in both cell culture supernatant and process intermediates was clearly underestimated by the Threshold system results when compared with the PicoGreen DNA assay results. The PicoGreen DNA assay clearly provides better accuracy and is a simpler procedure for measuring host cell DNA levels in cell culture supernatant and process intermediates than the conventional method with the Threshold system. This newly developed DNA assay will be prominent among host cell DNA assays for measuring host cell DNA levels in bio-pharmaceuticals.