Mast cells are essential in mediating the inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and beta-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, P14R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R2 > 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics, and to be a major tool of allergic drug assessment or anti-allergic drug development in research.