The ability to detect presence or absence of foreign gene(s) relies on high yields of pure DNA samples. Cotton seeds contain polyphenols, polysaccharides and other secondary metabolites which often interfere during DNA isolation. Here we present optimization of DNA isolation and Real Time PCR conditions for the detection of GMO in cotton seeds (Gossypium spp.). The modified CTAB protocol includes addition of sodium bisulfite in combination with higher CTAB concentration and precipitation of DNA with 5 M NaCl in combination with chilled ethanol increased the solubility of polysaccharides and polyphenols. This protocol is devoid of use of expensive chemicals such as Proteinase K and RNase etc. We also optimized Real Time PCR conditions such as annealing temperature and concentration of DNA for the detection of CaMV 35S target gene. Four samples out of five were found to be GM positive with reference to target gene.