The level of airway constriction in thin slices of lung tissue is highly variable. Owing to the labor-intensive nature of these experiments, determining the number of airways to be analyzed in order to allocate a reliable value of constriction in one mouse is challenging. Herein, a new automated device for physiology and image analysis was used to facilitate high throughput screening of airway constriction in lung slices. Airway constriction was first quantified in slices of lungs from male BALB/c mice with and without experimental asthma that were inflated with agarose through the trachea or trans-parenchymal injections. Random sampling simulations were then conducted to determine the number of airways required per mouse to quantify maximal constriction. The constriction of 45 ± 12 airways per mouse in 32 mice were analyzed. Mean maximal constriction was 37.4 ± 32.0%. The agarose inflating technique did not affect the methacholine response. However, the methacholine constriction was affected by experimental asthma (p = 0.003), shifting the methacholine concentration-response curve to the right, indicating a decreased sensitivity. Simulations then predicted that approximately 35, 16 and 29 airways per mouse are needed to quantify the maximal constriction mean, standard deviation and coefficient of variation, respectively; these numbers varying between mice and with experimental asthma.