Type I collagen is a major component of the extracellular matrix as well as many tissue engineered models. To understand changes in collagen related models over time, it is important to evaluate collagen dynamics with noninvasive techniques. Fluorescence spectroscopy provides a method to noninvasively measure endogenous collagen fluorescence. Additionally, second harmonic generation (SHG) imaging of collagen produces high resolution images of the fibrils. In this study, a novel in vitro collagen measurement chamber was developed for measurement in standard spectroscopic cuvette chambers and microscopic imaging. The fluorescence of polymerized collagen was found to be highly variable, primarily depending on incubation time after polymerization. Changes in fluorescence over time were consistent with increases at UVA excitation wavelengths (lambda ex = 360 nm) and decreases at UVC excitation wavelengths (lambda ex = 270 nm), suggesting changes in nonenzymatic association of the collagen fibrils. SHG imaging of the collagen suggested that a stable network formed during polymerization. Unlike the fluorescence emission, SHG images from the gels varied little with time suggesting that SHG is not as sensitive to cross-linking or fibril-fibril associated changes. The developed measurement system will allow further studies on the effect of enzymatic cleavages and structural alterations on collagen fluorescence and SHG.