Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased > 2-fold in whole, dissected lungs from acutely challenged mice compared to control mice (p = 1.8x10^-4). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared to adjacent, pathologically normal lung parenchyma (p = 0.03). MMP-2/9 activity detected by ACPP cleavage co-localized with gelatinase activity measured with in situ DQ gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen challenged mouse lungs. The results demonstrate that ACPPs, like the MMP2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.