A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry . Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.