Extracellular matrix (ECM) remodeling contributes to the pathogenic changes in chronic obstructive pulmonary disease (COPD) and is both complex and not well understood. Collagen I, a component of the ECM altered in COPD airways, has second harmonic generation (SHG) properties. The SHG signal is coherent, propagating both forward (F) (primarily organized/mature collagen fibrils) and backward (B) (primarily disorganized/immature collagen fibrils) parallel to the incident light. The F/B SHG ratio was used to determine the proportion of organized to disorganized collagen, with lower variation in F/B ratio between sampling regions within the same patient and between patients in the same disease group compared with analyzing F and B data alone. The F/B ratio was independent of laser power drift, regions analyzed within a tissue and tissue orientation during analysis. Using this method, we identified a significant difference in collagen organization in airway tissue between COPD and nondiseased. We have developed a robust optimization and calibration methodology that will allow direct comparison of data obtained at different times and from multiple microscopes, which is directly adaptable for use with other tissue types. We report a powerful new tool for advancing our understanding of pathological ECM remodeling that may uncover new therapeutic targets in the future.