Microfluidic systems are important for performing precise reagent manipulations and reducing material consumption in biological assays. However, optical detection methods limit analyses to fluorescent or UV-active compounds and traditional 2D fluidic designs have limited degrees of freedom. This article describes a microfluidic device that has three inputs and performs 2592 distinct enzyme reactions using only 150 μL of reagent with quantitative characterization. This article also introduces imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (iSAMDI-MS) to map reaction progress, by immobilization of the product onto the floor of the microfluidic channel, into an image that is used for calculating the Michaelis constant (K m). This approach expands the scope of imaging mass spectrometry, microfluidic detection strategies, and the design of high-throughput reaction systems.