Second Harmonic Generation (SHG) of collagen signals allows for the analysis of collagen structural changes throughout metastatic progression. The directionality of coherent SHG signals, measured through the ratio of the forward-propagating to backward propagating signal (F/B ratio), is affected by fibril diameter, spacing, and order versus disorder of fibril packing within a fiber. As tumors interact with their microenvironment and metastasize, it causes changes in these parameters, and concurrent changes in the F/B ratio. Specifically, the F/B ratio of breast tumors that are highly metastatic to the lymph nodes is significantly higher than those in tumors with restricted lymph node involvement. We utilized in vitro analysis of tumor cell motility through collagen gels of different microstructures, and hence different F/B ratios, to explore the relationship between collagen microstructures and metastatic capabilities of the tumor. By manipulating environmental factors of fibrillogenesis and biochemical factors of fiber composition we created methods of varying the average F/B ratio of the gel, with significant changes in fiber structure occurring as a result of alterations in incubation temperature and increasing type III collagen presence. A migration assay was performed using simultaneous SHG and fluorescent imaging to measure average penetration depth of human tumor cells into the gels of significantly different F/B ratios, with preliminary data demonstrating that cells penetrate deeper into gels of higher F/B ratio caused by lower type III collagen concentration. Determining the role of collagen structure in tumor cell motility will aid in the future prediction metastatic capabilities of a primary tumor.