Ampicillin is one of the ß-lactam antibiotics commonly used in livestock farming. Testing of these antibiotic residues in milk is mandatory. A simple and rapid liquid chromatographic (LC) method was developed for the analysis of ampicillin residues in various bovine milk samples, i. e., raw milk from two sources, commercially processed skim milk and pasteurized, homogenized milk with vitamin D added. Materials were deproteinized with trichloroacetic acid (TCA) and acetonitrile. After centrifuge, the clear supernatant was reacted with formaldehyde and TCA under heat to form fluorescence derivatives. Ampicillin derivatives were quantitatively analyzed using a reversed phase LC system with fluorescence detection. Average recoveries of ampicillin spiked at 5 to 20 ppb were >85% with coefficients of variation <10%. Limits of detection ranged from 0.31 to 0.51 ppb and limits of quantitation ranged from 0.66 to 1.2 ppb for all four types of milk. Upon appropriate validation, this method is applicable for rapid determination of ampicillin residues in milk at the tolerance level of 10 ppb.