Background: Allergic sensitization is commonly assessed in patients by performing the skin prick test (SPT) or determining specific IgE levels in blood samples with the ImmunoCAP assay, which measures each allergen and sample separately. This paper explores the possibility to investigate respiratory allergies with a high throughput method, the Meso Scale Discovery (MSD) multiplex immunoassay, measuring IgE levels in low volumes of blood. Methods: The MSD multiplex immunoassay, developed and optimized with standards and allergens from Radim, was validated against the SPT and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients and 3 controls), blood collection and the SPT were performed within the same hour. Results: Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay and the ImmunoCAP assay, except for house dust mite. The sensitivity of the MSD multiplexed assay was ≥75% for most allergens compared to the SPT and ImmunoCAP assay. Additionally, the specificity of the MSD multiplex immunoassay was ≥80% - the majority showing 100% specificity. Only the rye allergen had a low specificity when compared to the SPT, probably due to cross-reactivity. The reproducibility of the MSD multiplex immunoassay, assessed as intra- and inter-assay reproducibility and biological variability between different sampling moments, showed significantly high correlations (r=0.943-1) for all tested subjects (apart from subject 13; r=0.65-0.99). Conclusion: The MSD multiplex immunoassay is a reliable method to detect specific IgE levels against respiratory allergens in a multiplexed and high throughput way, using blood samples as small as from a finger prick.