Blotting is the key technique of current molecular biology, and it is an efficient and a sensitive way of determining if a sample contains a certain biomolecule. The array of technique known as “Southern blotting,” in which a particular restriction fragment can be recognized against the background of more restriction fragments. The process comprises transferring DNA fragments from the electrophoresis gel onto a nitrocellulose membrane in a way that evoke the gel’s DNA banding pattern. After that, hybridization probing takes place to detect the desired restriction fragment. To investigate gene targets, Southern blotting is frequently used as the benchmark for identifying transgenic integration and effective homologous recombination. This chapter enlightens the following various steps to perform the Southern blotting that includes genomic DNA isolation, restriction digestion of isolated DNA, agarose gel electrophoresis, depurination, denaturation, neutralization then transfer of DNA from agarose gel to nylon membrane (blotting), pre-hybridization, probe preparation and random labeling of DNA fragments, hybridization, and post-hybridization (washing) and exposed the x-ray film on the membrane and autoradiography or scanning to visualize the hybridized DNA fragments.