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3D paper-based cultures (PBCs) are easy-to-use and provide a biologically representative microenvironment. By stacking a sheet of cell-laden paper below sheets containing cell-free hydrogel, we form an assay capable of segmenting cells by the distance they invaded from the original cell-seeded layer. These invasion assays are limited to end-point analyses with fluorescence-based readouts due to the highly scattering nature of the paper scaffolds. Here we demonstrate that optical coherence tomography (OCT) can distinguish living cells from the surrounding extracellular matrix (ECM) or paper fibers based upon their intracellular motility amplitude (M).Mis computed from fluctuation statistics of the sample, rejects shot noise, and is invariant to OCT signal attenuation. Using OCT motility analysis, we tracked the invasion of breast cancer cells over a 3-day period in 4-layer PBCs (160–300 µm thick)in situ. The cell population distributions determined with OCT are highly correlated with those obtained by fluorescence imaging, with an intraclass correlation coefficient (ICC) of 0.903. The ability of OCT motility analysis to visualize live cells and quantify cell distributions in PBC assaysin situand longitudinally provides a novel means for understanding how chemical gradients within the tumor microenvironment affect cellular invasion.