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This paper describes a method that combines a microfluidic device and self‐assembled monolayers for matrix‐assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self‐assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca2+. Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K0.5) for the Ca2+ – dependent activation of PAD2. This work establishes a high‐throughput and label‐free method for studying enzyme‐ligand binding interactions and widens the applicability of microfluidics and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry. [ABSTRACT FROM AUTHOR]