Electrospray ionisation mass spectrometry (ESI-MS) has been used for the determination and quantitation of a broad range of 24 antibiotics, from groups including aminoglycosides, beta -lactams, tetracyclines, antifungals and glycopeptides. Spectra have been acquired for all 24 antibiotics derived from pure samples dissolved in acetonitrile/water, along with samples extracted from complex fermentation liquor. Quantitation was carried out by the detection of the protonated molecules, using time-scheduled single-ion monitoring (SIM). ESI-MS was used to detect and quantify to 5-muM levels. A one-step extraction of antibiotics with an organic solvent (methanol) was used for this rapid and simple procedure. Specificity is not matched by other methods and antibiotic analogues (e.g. the five forms of erythromycin) can be determined within minutes. Copyright (C) 2001 John Wiley & Sons, Ltd.