A quantitative method to assess relative potencies (IC 50) of 5-lipoxygenase (5-LO) enzyme inhibitors was established in antigen-induced contractions of tracheas isolated from actively sensitized guinea pigs (Schultz-Dale model). The relative potencies of four purported 5-LO inhibitors determined in this tissue assay were compared with those from a crude enzyme preparation isolated from guinea pig neutrophils. All compounds suppressed ovalbumin (OA)-induced tracheal contractions in a concentration-related manner in the presence of indomethacin and pyrilamine. IC 50 Values, determined from the percent inhibition values obtained from responses at 30 ng/mL OA of these compounds ranged from 0.56–15 μM. A similar rank order of potency for inhibition of 5-HETE formation from a crude enzyme preparation was observed. This suggested that these agents had a common mechanism of action in the two assay systems and further validated the IC 50 values determined in trachea assay. LY171883, an LTD 4/LTE 4 receptor antagonist, also suppressed OA-induced contractions concentration dependently with an IC 50 of 4.9 μM determined by this method. LTD 4 concentration-response curves were not altered by any of the four 5-LO inhibitors, ruling out the possibility that these agents were acting as LT receptor antagonists. Results of this study demonstrated that relative potencies of 5-LO inhibitors can be quantitatively assessed using this airway tissue model, which helps in identifying potential therapeutic agents for asthma.