BACKGROUND Treatment effects in allergen immunotherapy (AIT) studies are based on symptomatic improvement, and evaluations of naturally exposed patients do often show weak efficacy. Allergen challenge tests, such as conjunctival (CAC), nasal (NAC), or bronchial (BAC) challenge tests, or challenges in allergen exposure chambers (AEC) are accepted by regulators for AIT phase II studies only. METHODS This review aims to describe different allergen challenge test methods, summarizes safety and limitations for each, and discusses their potential for use in AIT trials. RESULTS Organ-specific allergen challenges provide information about individual reactivity, reaction threshold, and organ-specific efficacy of AIT. AECs, targeting all affected organs simultaneously, were developed to investigate disease mechanisms and treatment effects under controlled and reproducible conditions. CONCLUSIONS A high level of standardization is existing for NAC only; in CAC and BAC, the toolbox is limited to subjective symptom scoring with no validated objective parameters identified yet. AECs are complex and heterogenous; correlation of systems and comparability of study data is claimed. All challenge methods are safe when conducted by experienced staff.