Determination of caseins in food matrices is usually performed by using the competitiveenzyme- linked immunosorbent assay (ELISA) technique. However such a technique suffers from a number of limitations. Among these, the applicability to a narrow concentration range, a non linear (logarithmic) response, a non-negligible cross-reactivity and a high cost per kit. At the time of the completion of this study, in case of ELISA positive feedback, there was poor availability in the literature of finding reliable instrumental methods able to determine both qualitatively and quantitatively this class of substances. In the present study, a liquid chromatography-high resolution tandem mass spectrometry (HPLC-HRMS/MS) instrumental method was developed with a high resolution mass spectrometer (Orbitrap). Real samples of sausages in which caseins were detected by ELISA technique were analysed. A casein-free sample of ham was used as a blank. The analytical characteristics of the instrumental method were compared with those of a commercial ELISA test, declared specific for α- and β-casein.