The subepithelial thickening of asthmatic airways is manifested by increased fibroblast/myofibroblast proliferation and deposition of extracellular matrix proteins including collagen. This feature of asthma can be modeled in mice that are sensitized and challenged with ovalbumin, but quantitation has been suboptimal. In this study, we describe the development of a quantitation method using computerized image analysis. Mice (SV129/C57BL/6) were sensitized by intraperitoneal injection of 10 μg of ovalbumin in 0.1 mL of saline solution on two occasions 10 days apart. Twenty-one days after the second sensitization, mice were challenged by intratracheal instillation of 400 μg of ovalbumin in 50 μL of saline solution daily for 6 days. Control groups included ovalbumin-sensitized/sham-challenged, sham-sensitized/ovalbumin-challenged, and sham-sensitized/sham-challenged mice. Each group contained from 7 to 11 mice. Twelve days after the final challenge, the lungs were perfused, fixed via the vasculature, and inflated with paraformaldehyde at a pressure of 20 cm H2O. Transverse sections of lung (3 μm) were stained using a modified Martius scarlet blue technique, and subepithelial collagen staining was quantitated using a computer-assisted image analysis system. Airways were examined using color thresholding for airspace, pixels were converted into micrometers, and lumen perimeter was measured. Airways were then examined using color thresholding for collagen staining. Subepithelial collagen, staining blue, was selected, and its area was measured. Eight to 16 airways per animal were analyzed, and results were expressed as mean area (micrometers squared) of collagen per micrometer of airway perimeter. There were no significant differences in collagen deposition between the control groups (ovalbumin-sensitized/sham-challenged group, 5.65 ± 0.26; sham-sensitized/ovalbumin-challenged group, 5.63 ± 0.27; sham-sensitized/sham-challenged group, 6.31 ± 0.32). Ovalbumin sensitization and challenge produced significantly more subepithelial collagen deposition than each of the control treatments, 7.77 ± 0.37 (p < 0.01 in all cases). This represented an increase of 33% in subepithelial collagen deposition compared with the mean of the three control groups. Results were confirmed by an independent observer. We conclude that airway subepithelial collagen can be accurately and reproducibly measured using computerized image analysis in mice. This methodology will have applications in studies of mechanisms involved in and assessment of interventions to modulate airway remodeling.