The quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine β-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from β-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured β-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg– 1 with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%). Reprinted from Food Additives & Contaminants: Part A. 32: 1 15 December 2014.