Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.