A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.