The introduced electrospray (ESP) technique combined with quadrupole mass spectrometry (MS) was applied for the trace residue detection (10 ppb) of commonly administered beta-lactam antibiotics in bovine milk. Because of the widespread use of penicillin G, ampicillin, amoxicillin, cephapirin, cloxacillin, and ceftiofur in veterinary medicine, these six popular drugs were chosen to develop a fast and reliable microcolumn liquid chromatographic (micro-LC) separation method for residue-containing milk extracts. Furthermore, the analytes were selected to test the chromatographic behavior of a novel stationary phase, a perfusive-particle column packed with derivatized porous polystyrene divinylbenzene. The effects of mobile phase additives on separation and ESP ionization efficiency were investigated. The ionization of the eluted analytes took place either in a conventional ESP interface with a capillary shield allowing flow rates up to 40 microL/min or in the latest model of the ultrasonic nebulization ESP interface. The perfusive-particle column, although not optimized for the separation of small molecules, exhibited sufficient resolution of the antibiotics for its routine usage, especially when considering the remarkable time-saving advantage compared with ordinary reversed phase micro-LC columns. The application of the novel ultrasonic nebulization interface lowered detection limits and improved the stability of the signals over a much wider flow rate range than possible for ionization with the ESP system equipped with the conventional needle.