Reversible airway obstruction and airway inflammation following allergen exposure represent crucial pathophysiological features of allergic asthma. In order to investigate these events, bronchial-specific challenge is commonly employed although it may be complicated and may not be free of possible risks. Therefore, we evaluated the applicability of allergen-specific conjunctival challenge (ASCC) in adults with asthma to define the diagnosis of specific sensitization and the grade of specific sensitization, and to detect the presence of allergic inflammation. As recently described, conjunctival challenge represents an easy diagnostic tool in allergic inflammation. We studied 30 symptomless patients with a history of mild asthma, including 20 allergic patients, 10 females and 10 males (18-48 years old), sensitized to Parietaria judaica only, with a history of recurrent asthma attacks during seasonal natural exposure to allergen, 10 nonallergic patients, 7 females and 3 males (28-55 years old), with a history of recurrent asthma attacks unrelated to any allergen exposure, and 10 healthy volunteers as controls. The study was performed outside the pollen season. The ASCC induced a highly significant early-phase clinical reaction and a highly significant inflammatory cell infiltrate in the asthmatic patients sensitized to P. judaica when compared to other study groups (p < 0.001). The ASCC showed as 100% sensitivity, specificity, predictive value of positive and negative results in the group of sensitized asthmatic patients when compared to unsensitized ones and to healthy subjects. The study confirms the reproducibility and usefulness of ASCC to diagnose allergy and to define its grade. These data strongly suggest ASCC employment also in patients with a single history of allergic asthma.