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Cell migration is essential in many physiological and pathological processes. To understand this complex behavior, researchers have turned to quantitative, in vitro, image-based measurements to dissect the steps of cellular motility. With the rise of automated microscopy, the bottleneck in these approaches is no longer data acquisition, but data analysis. Using time-lapse microscopy and computer-assisted image analysis, we have developed a novel, quantitative assay that extracts a multivariate profile for cellular motility. This technique measures three dynamic parameters per single cell: speed, surface area, and an in-dex of cell expansion/contraction activity (DECCA). Our assay can be used in combination with a variety of extracellular matrix components, or other soluble agents, to analyze the effects of the microenvironment on cellular migration dynamics in vitro. Our application was developed and tested using A431 and HT-1080 cell lines plated on laminin-332 or fibronectin substrates. Our results indicate that HT-1080 cells migrate faster, have a greater surface area, and have a higher DECCA index than A431 cells on both matrices (for all parameters, p < 0.05). Spearman's correlation coefficients suggest that for these cell lines and matrices, various combinations of the three measurements display low to medium-high levels of correlation. These findings compare well with previous literature. Our approach provides new tools to measure cellular migration dynamics and address questions on the relationship between cell motility and the microenvironment, using only common microscopy techniques, accessible image analysis applications, and a basic desktop computer for image processing.