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Background. Food allergy is an increasing public health issue and the commonest cause of life-threatening anaphylactic reactions. Conventional allergy tests assess for the presence of allergen-specific IgE, significantly overestimating the rate of true clinical allergy resulting in over-diagnosis and adverse impact on health-related quality of life. Objective. To undertake initial validation and assessment of a novel diagnostic tool, the mast cell activation test (MAT). Methods. Primary human mast cells (hMCs) were generated from peripheral blood precursors, and sensitized using patient sera and then incubated with allergen. Mast cell degranulation was assessed by flow cytometry and mediator release. We compared the diagnostic performance of MAT to existing diagnostic tools to assess in a cohort of peanut-sensitized individuals undergoing double-blind, placebo-controlled challenge. Results. hMCs sensitized with sera from peanut, grass pollen and hymenoptera- (wasp venom) allergic patients demonstrated allergen-specific and dose-dependent degranulation by both expression of surface activation markers (CD63 and CD107a) and functional assays (prostaglandins D2 and ß-hexosaminidase release). In this cohort of peanut-sensitized individuals, MAT was found to have superior discrimination performance compared to other testing modalities including component-resolved diagnostics and basophil activation test. Using functional principle component analysis, we identified 5 clusters or patterns of reactivity in the resulting dose-response curves, which at preliminary analysis corresponded to the reaction phenotypes seen at challenge. Conclusion. MAT is a robust tool which may confer superior diagnostic performance compared to existing allergy diagnostics, and may be useful to explore differences in effector cell function between basophils and mast cells during allergic reactions.