Chemotaxis has been shown to be beneficial for the migration of soil-inhabiting bacteria towards industrial chemical pollutants, which they degrade. Many studies have demonstrated the importance of this microbial property under various circumstances; however, few quantitative analyses have been undertaken to measure the two essential parameters that characterize the chemotaxis of bioremediation bacteria: the chemotactic sensitivity coefficient 0 and the chemotactic receptor constant K-c. The main challenge to determine these parameters is that 0 and K-c are coupled together in non-linear mathematical models used to evaluate them. In this study we developed a method to accurately measure these parameters for Pseudomonas putida in the presence of toluene, an important pollutant in groundwater contamination. Our approach uses a multilayer microfluidic device to expose bacteria to a convection-free linear chemical gradient of toluene that is stable over time. The bacterial distribution within the gradient is measured in terms of fluorescence intensity, and is then used to fit the parameters K-c and 0 with mathematical models. Critically, bacterial distributions under chemical gradients at two different concentrations were used to solve for both parameters independently. To validate the approach, the chemotaxis parameters of Escherichia coli strains towards -methylaspartate were experimentally derived and were found to be consistent with published results from related work. Biotechnol. Bioeng. 2015;112: 896-904. (c) 2014 Wiley Periodicals, Inc.