Aims: To establish a new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins. Methods and results: Liquid chromatography/mass spectrometry (LC/MS) was employed for development of a new system for rapid detection and identification of bacteriocins. The system detected and identified bacteriocins such as nisin and lacticin 481 from 25 mu l of culture supernatants of their producing strains by accurate mass determination coupled with simultaneous impurity removal within 40 min. Especially, the system clearly distinguished three nisin variants (A, Z, Q) in culture supernatants of their producing strains, although they have similar structures and molecular masses. Each one-step pretreatment by cell adsorption-desorption or acetone precipitation improved bacteriocin detection dramatically, especially for mundticin KS. This system could be applied for detection and molecular mass determination of novel bacteriocins by extracting bacteriocin-related ions. Conclusions: The developed system could detect and identify some kinds of bacteriocin from culture supernatants or pretreated samples. Significance and Impact of the Study: The developed system helps us to identify bacteriocins in the early stage of screening without any or with one-step pretreatment. This system is effective on not only detection of known bacteriocins but also identification of novel bacteriocins. Consequently, this system will accelerate discovery of novel bacteriocins.