A screening method based on liquid chromatography/electrospray tandem mass spectrometry was developed in order to control the illegal use of corticosteroids as growth promoters in cattle. The objective was the detection of low residue levels of corticosteroids or metabolites in biological matrices. Relative to other studies published on this subject, the present work focused on enhancing specificity and sensitivity. Firstly, fragmentation of corticosteroids by collision-induced dissociation was studied. In positive mode, the losses of H2O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds, were observed. For higher collision energy, fragmentations in the B, C and D rings were induced. The negative mode was found to be more specific, inducing a cleavage of the C20–C21 bond with concomitant loss of formaldehyde (CH2O). Secondly, three acquisition methods in the negative mode were studied and evaluated, recorded signals being the parent ion [M ‡ acetate]ÿ and the two daughter ions, [M ÿ H]ÿ and [M ÿ H ÿ CH2O]ÿ. For dexamethasone, MS/MS instrumental detection limits of fragment ion and neutral loss scans, and of multiple reaction monitoring (MRM), were 250, 20 and 5 pg injected, respectively. The MRM method was then evaluated with the objective of use for the detection of corticosteroid residues in biological samples (urine, hair, muscle) and for a metabolism study.