beta 2-Agonist drugs may be illegally used as growth promoters for feedlot calves, when mixed into milk replacer immediately before feeding. To check for the presence of clenbuterol, salbutamol and terbutaline in such food, an analytical system was established using a screening method based on two commercial qualitative competitive ELISA tests, with antibodies raised against the arylamino group and the t-butyl group. The extraction procedure was based on precipitation of the milk samples with acetonitrile followed by filtration. The absence of any significant interference by other substances in the filtrate allowed detection of beta 2-agonist drugs in spiked samples at the lowest concentration having a repartitioning effect (50 ppb for clenbuterol, mabuterol and terbutaline, 500 ppb for salbutamol). In view of a false positive response with tetracycline in milk samples and a cross-reaction between clenbuterol and mabuterol, an HPLC-MS technique was developed which, after extraction and purification of the samples with SPE C18 Polar Plus, was able to confirm the presence of these drugs. The good recovery after extraction (ranging from 84% to 90.2%) and the low detection limit with this method (250 ng/ml for clenbuterol, mabuterol and terbutaline, and 2.5 micrograms/ml for salbutamol) allowed easy confirmation and simultaneous detection of the four beta 2-agonists at the lowest concentrations at which they are used in adulterated milk for calves.