In prostate cancer, bone is a frequent site of metastasis; however, the molecular mechanisms of this tumor tropism remain unclear. Here, we integrate a microfluidic coculture platform with multi-photon imaging based techniques to assess both phenotypic cell behavior and FAD fluorescence intensity and fluorescence lifetime in the same cell. This platform combines two independent assays normally performed with two different cell populations into a single device, allowing us to simultaneously assess both phenotypic cell behavior and enzyme activity. We observed that the osteotropic prostate cancer cell line (C4-2B), when in a coculture with bone marrow stromal cells (MC3T3-E1), has increased protrusive phenotype and increased total and protein-bound FAD compared to its parent cell line (LNCaP). We hypothesized that an increase in ROS-generating APAO activity may be responsible for these effects, and found that the effects were decreased in the presence of the antioxidant N-Acetyl Cysteine (NAC). This suggests that an ROS-related signaling mechanism at the bone metastatic site may be correlated with and play a role in increased invasion of metastasizing prostate cancer cells. The studies performed using this combined platform will lead to new insights into the mechanisms that drive prostate cancer metastasis.