Background: In tree nut (TN) allergy, singleplex tests showed the diagnostic utility of rAna o 3, rCor a 14/nCor a 9, and nJug r 1/nJug r 4 for cashew/pistachio, hazelnut, and walnut allergies, respectively. However, disadvantages of the tests include high costs and excessive blood sampling in multi-sensitized patients, and a limited number of components. We investigated the utility of a multiplex macroarray (i.e., the ALEX2 test) in TN allergy. Methods: In 169 children, skin prick test, the component- and extract-specific IgEs of TNs were investigated for clinical reactivity and tolerance. Results: The predictors (AUC = 0.962-0.749) of clinical reactivity to cashew, pistachio, hazelnut, and walnut were rPis v 1/rAna o 3, rPis v 1/rAna o 3/nPis v 2/nPis v 3, rCor a 14/nCor a 11/nCor a 9, and nJug r 1/nJug r 2/nJug r 6/nJug r 4, respectively. More than 93% of the patients with clinical reactivity to pistachio/cashew, hazelnut and walnut had positivity of (>= 0.3 kU(A)/L) rPis v 1/rAna o 3, rCor a 14 and nJug r 1/nJug r 2, respectively. The highest accuracies of clinical reactivity to culprit nut were obtained with combination of rPis v 1, sIgE and SPT positivities for cashew/pistachio, rPis v 1 >= 1.0 kU(A)/L for pistachio, rCor a 14 >= 1.0 kU(A)/L for hazelnut and combination of nJug r 1 and nJug r 2 positivities for walnut, respectively. Also, higher concentrations of rPis v 1 (>= 15.0 kU(A)/L), rCor a 14 (>= 5.0 kU(A)/L) and nJug r 1/nJug r 2 (>= 15.0 kU(A)/L) had %100 specificity and PPV in predicting clinical reactivity to cashew, hazelnut and walnut, respectively. Conclusions: Multiplex macroarray test is useful and reliable in the diagnosis of TN allergy in children, confirms and expands existing knowledge, and can be used as a stand-alone tool in the bottom-up diagnostic approach. Copyright (c) 2021, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).