The plug-in-pond and capillary assays are convenient methods for measuring attractant and repellent bacterial chemotaxis. However, these assays do not provide quantitative information on the extent of migration and are not well-suited for investigating repellent taxis. Here, we describe a protocol for a flow-based microfluidic system (μFlow) to quantitatively investigate chemotaxis in response to concentration gradients of attractants and repellents. The μFlow device uses diffusive mixing to generate concentration gradients that are stable throughout the chemotaxis chamber and for the duration of the experiment. The gradients may be of any desired absolute concentration and gradient strength. GFP-expressing bacteria immediately encounter a stable concentration gradient when they enter the chemotaxis chamber, and the migration in response to the gradient is monitored by microscopy. The effects of different parameters that influence the extent of migration in the μFlow device—preparation of the motile bacterial population preparation, strength of the concentration gradient and duration of exposure to the gradient—are discussed in the context of repellent taxis of chemotactically wild-type Escherichia coli cells in a gradient of NiSO₄. Fabrication of the microfluidic device takes 1 d while preparing motile cells and carrying out the chemotaxis experiment takes 4–6 h to complete.