The antagonistic activity is normally assayed by the agar-well diffusion method under aerobic conditions. Pre-poured agar media plates are spread with appropriate dilution i.e. cfu of the respective indicator microorganism and allow to dry. In the agar plates, wells of 5 mm diameter are cut using a cork borer. The wells are filled with few known volume in μl of the cell-free supernatant of organism. The antagonistic zones are detected after incubating for 24–72 h depending on the species of microorganisms at 30 °C.

Using   indicator microorganism, the antagonistic activity of the sample are tested by few known fold serial dilutions. The antagonistic activity is expressed in terms of arbitrary units (AU). It is defined as the maximum dilution produced a minimum of zone that still gives a clearly visible antagonistic zone. The reciprocal of the dilution give the titer of antagonistic activity in AU per milliliter.