Many chemotaxis assays allow for the assessment of bacterial chemotaxis by determining the number of cells migrating toward a chemoattractant or away from a chemorepellent. Some of these assays use a capillary filled with a chemoeffector/agarose mixture to allow cells to accumulate at the mouth of the capillary. Subsequently, assumptions about the relative strengths of chemotaxis strength are based on visual comparisons. Here, we describe a modification of this assay that uses a hydrogel matrix to enable quantitative time-course measurements by analyzing image pixel intensities. This approach allows a high-throughput method when coupled with the aid of a motorized microscope stage.