Although intradermal testing (IDT) is commonly used in the etiological diagnosis of allergies, in vitro testing for specific IgE (sIgE) is an attractive alternative. Currently, new laboratory techniques in veterinary allergological practice, including multiple allergen simultaneous tests (MASTs), gradually supersede in vivo tests. Both, serological (sIgE) and IDTs in fourteen atopic Malopolski horses were performed. Correlation and agreement between test results were evaluated. Receiver operating characteristic analysis showed that sIgE to Acarus siro had the best diagnostic performance (Area under the ROC curve [AUC] = 0.969), followed by Dermatophagoides pteronyssinus (AUC = 0.844), Dermatophagoides farinae (AUC = 0.813) and Tyrophagus putrescentiae (AUC = 0.803). A significant positive correlation between IDT and MAST was found for A. siro (r S = 0.870; p = 0.00005), and D. farinae (r S = 0.657; p = 0.011). There was significant moderate agreement for 2 of 5 allergens, A. siro (κ = 0.569) and D. farinae (κ = 0.485) in semiquantitative assessment and significant fair to substantial agreement for 3 of 5 allergens, D. pteronyssinus (κ = 0.689), A. siro (κ = 0.569), D. farinae (κ = 0.432) in dichotomic assessment. Sensitivity ranged from 44% to 89%, depending on the allergen, while specificity was significantly higher for all allergens in MAST (60%-100%); the mean accuracy was 73% (manufacturer cutoff) and 77.4% (optimal cutoff) based on the Youden index. Compared with IDT, serological MAST showed good detection performance for 60% allergen sIgE in dichotomic assessment with substantial diagnostic capability, but careful clinical interpretation is needed for some allergens.